Compositions and methods for inhibiting expression of Mylip/Idol gene

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the Mylip/Idol gene, and methods of using such dsRNA compositions to inhibit expression of Mylip/Idol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Entry Application ofInternational Application No. PCT/US2011/22339, filed Jan. 25, 2011,which designates the United States, and which claims benefit under 35U.S.C. §119(e) of U.S. provisional application 61/297,954 filed on Jan.25, 2010, the contents of which are incorporated herein in theirentireties by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 23, 2012, isnamed 20120725_SequenceListing-TextFile_(—)051058_(—)054000_US.txt andis 373,982 bytes in size.

FIELD OF THE INVENTION

The invention relates to the specific inhibition of the expression ofthe Mylip/Idol gene.

BACKGROUND OF THE INVENTION

Myosin Regulatory light chain interacting protein (Mylip) is an ERM-likeprotein that interacts with myosin regulatory light chain and inhibitsneurite outgrowth in neurons. The Mylip protein comprises a FERMhomology domain at the N-terminus, and a RING zinc finger ubiquitinligase domain at the C-terminus. While FERM-containing proteins areknown to interact with the cytoplasmic regions of transmembraneproteins, Mylip is presently the only FERM-containing protein known tointeract with the myosin regulatory light chain protein. Mylip isexpressed ubiquitously in almost all human tissues.

Mylip has been shown to downregulate the LDL receptor by enhancing LDLreceptor ubiquitination and leading to degradation of the LDL receptor(LDL-R). Overexpression of the Mylip protein in mice reduces levels ofthe LDL-R, decreases LDL uptake into cells and increases plasmacholesterol levels. Conversely, inhibition of Mylip expression in miceenhances LDL uptake into cells. Given the actions of Mylip on LDL-Rexpression, the protein is also referred to as ‘inducible degrador ofthe LDL-R’ (Idol).

Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseasesin which patients exhibit elevated total and LDL cholesterol levels,tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J.Clin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessiveform, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C.,(2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDLuptake by the liver. ADH may be caused by LDLR mutations, which preventLDL uptake, or by mutations in the protein on LDL, apolipoprotein B,which binds to the LDLR. ARH is caused by mutations in the ARH proteinthat are necessary for endocytosis of the LDLR-LDL complex via itsinteraction with clathrin. As Mylip/Idol plays a role inreceptor-mediated LDL uptake it is likely that treatment strategiesdirected at Mylip/Idol would be beneficial in the above-describeddisorders.

SUMMARY OF THE INVENTION

Described herein are compositions and methods that effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of the Mylip/Idol gene, such as in a cell or mammal. Alsodescribed are compositions and methods for treating pathologicalconditions and diseases caused by the expression of a Mylip/Idol gene,such as a lipid disorder or metabolic disorder (e.g., atherosclerosis ordiabetes). Also described are compositions and methods described forpromoting neurite outgrowth, thus permitting treatment ofneurodegenerative disorders or nerve damage such as e.g., spinal cordinjury.

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein inhibits the expressionof Mylip/Idol in a cell or mammal. Alternatively, in another embodiment,the iRNA up-regulates the expression of Mylip/Idol in a cell or mammal.

The iRNAs included in the compositions featured herein encompass a dsRNAhaving an RNA strand (the antisense strand) having a region that is 30nucleotides or less, generally 19-24 nucleotides in length, that issubstantially complementary to at least part of an mRNA transcript of aMylip/Idol gene. In one embodiment, the dsRNA comprises a region of atleast 15 contiguous nucleotides.

In one embodiment, an iRNA for inhibiting expression of a Mylip/Idolgene includes at least two sequences that are complementary to eachother. The iRNA includes a sense strand having a first sequence and anantisense strand having a second sequence. The antisense strand includesa nucleotide sequence that is substantially complementary to at leastpart of an mRNA encoding Mylip/Idol, and the region of complementarityis 30 nucleotides or less, and at least 15 nucleotides in length.Generally, the iRNA is 19 to 24, e.g., 19 to 21 nucleotides in length.In some embodiments the iRNA is from about 15 to about 25 nucleotides inlength, and in other embodiments the iRNA is from about 25 to about 30nucleotides in length. The iRNA, upon contacting with a cell expressingMylip/Idol, inhibits the expression of a Mylip/Idol gene by at least10%, at least 20%, at least 25%, at least 30%, at least 35% or at least40% or more, such as when assayed by a method as described herein. Inone embodiment, the Mylip/Idol iRNA is formulated in a stable nucleicacid lipid particle (SNALP).

In one embodiment, an iRNA featured herein includes a first sequence ofa dsRNA that is selected from the group consisting of the sensesequences of Tables 3, 4, 5 and 6, and a second sequence that isselected from the group consisting of the corresponding antisensesequences of Tables 3, 4, 5 and 6. The iRNA molecules featured hereincan include naturally occurring nucleotides or can include at least onemodified nucleotide, including, but not limited to a 2′-O-methylmodified nucleotide, a nucleotide having a 5′-phosphorothioate group,and a terminal nucleotide linked to a cholesteryl derivative.Alternatively, the modified nucleotide may be chosen from the group of:a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.Generally, such a modified sequence will be based on a first sequence ofsaid iRNA selected from the group consisting of the sense sequences ofTables 3, 4, 5 and 6, and a second sequence selected from the groupconsisting of the antisense sequences of Tables 3, 4, 5 and 6.

In another embodiment, a composition containing a dsRNA targetingMylip/Idol is administered to a subject when Low Density Lipoproteincholesterol (LDLc) levels reach or surpass a predetermined minimallevel, such as greater than 130 mg/dL, 150 mg/dL, 200 mg/dL, 300 mg/dL,or 400 mg/dL. In another embodiment, the subject has an LDLc levelgreater than about 150 mg/dL.

In one embodiment, a single administration of the dsRNA lowers LDLclevels by at least 10%, e.g., by at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, or at least 60%, or more.In another embodiment, the lowered LDLc level is maintained for at least5, 10, 20, 30, or 40 days or longer.

In one embodiment, the subject is selected, at least in part, on thebasis of needing (as opposed to merely selecting a patient on thegrounds of who happens to be in need of) LDL lowering, LDL loweringwithout lowering of HDL, ApoB lowering, or total cholesterol loweringwithout HDL lowering.

In one embodiment, an iRNA as described herein targets a wildtypeMylip/Idol RNA transcript, and in another embodiment, the iRNA targets amutant transcript (e.g., a Mylip/Idol RNA carrying an allelic variant).For example, an iRNA of the invention can target a polymorphic variant,such as a single nucleotide polymorphism (SNP), of Mylip/Idol. Inanother embodiment, the iRNA targets both a wildtype and a mutantMylip/Idol transcript. In yet another embodiment, the iRNA targets atranscript variant of Mylip/Idol.

In one embodiment, an iRNA featured in the invention targets anon-coding region of a Mylip/Idol RNA transcript, such as the 5′ or 3′untranslated region.

In one aspect, embodiments of the invention provide a cell containing atleast one of the iRNAs featured in the invention. The cell is generallya mammalian cell, such as a human cell.

In another aspect, embodiments of the invention provide a pharmaceuticalcomposition for inhibiting the expression of a Mylip/Idol gene in anorganism, generally a human subject. The composition typically includesone or more of the iRNAs described herein and a pharmaceuticallyacceptable carrier or delivery vehicle. In one embodiment, thecomposition is used for treating a lipid disorder, such asatherosclerosis. In another embodiment, the composition is used fortreating a spinal cord injury or a neurodegenerative disease ordisorder, such as palsy, or Parkinson's disease.

In another embodiment, the pharmaceutical composition is formulated foradministration of a dosage regimen described herein, e.g., not more thanonce every four weeks, not more than once every three weeks, not morethan once every two weeks, or not more than once every week. In anotherembodiment, the administration of the pharmaceutical composition can bemaintained for a month or longer, e.g., one, two, three, or six months,or one year, or five years, or ten years, or longer, including theremaining lifetime of a subject.

In another embodiment, a composition containing an iRNA describedherein, e.g., a dsRNA targeting Mylip/Idol, is administered with anon-iRNA therapeutic agent, such as an agent known to treat a lipiddisorder, or a symptom of a lipid disorder. For example, an iRNAfeatured in the invention can be administered with an agent fortreatment of atherosclerosis or hypercholesterolemia or other disordersassociated with cholesterol metabolism.

In another embodiment, a Mylip/Idol iRNA is administered to a patient,and then the non-iRNA agent is administered to the patient (or viceversa). In another embodiment, a Mylip/Idol iRNA and the non-iRNAtherapeutic agent are administered at the same time. In one embodiment,the agent is, for example, an agent that affects cholesterol metabolism,such as an HMG-CoA reductase inhibitor (e.g., a statin).

In another aspect, provided herein is a method for inhibiting theexpression of a Mylip/Idol gene in a cell by performing the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA includes at least two sequences that        are complementary to each other. The dsRNA has a sense strand        having a first sequence and an antisense strand having a second        sequence; the antisense strand has a region of complementarity        that is substantially complementary to at least a part of an        mRNA encoding Mylip/Idol, and where the region of        complementarity is 30 nucleotides or less, i.e., 15-30        nucleotides in length, and generally 19-24 nucleotides in        length, and where the dsRNA, upon contact with a cell expressing        Mylip/Idol, inhibits expression of a Mylip/Idol gene by at least        10%, preferably at least 20%, at least 30%, at least 40% or        more; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        Mylip/Idol gene, thereby inhibiting expression of a Mylip/Idol        gene in the cell.

In another aspect, the invention provides methods and compositionsuseful for activating expression of a Mylip/Idol gene in a cell ormammal.

In another aspect, the invention provides a method for modulating theexpression of a Mylip/Idol gene in a cell by performing the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA includes at least two sequences that        are complementary to each other. The dsRNA has a sense strand        having a first sequence and an antisense strand having a second        sequence; the antisense strand has a region of complementarity        that is substantially complementary to at least a part of an        mRNA encoding Mylip/Idol, and where the region of        complementarity is 30 nucleotides or less, i.e., 15-30        nucleotides in length, and generally 19-24 nucleotides in        length, and where the dsRNA, upon contact with a cell expressing        Mylip/Idol, modulates expression of a Mylip/Idol gene by at        least 10%, preferably at least 20%, at least 30%, at least 40%        or more; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation or protection of the mRNA        transcript of the Mylip/Idol gene, thereby modulating expression        of a Mylip/Idol gene in the cell.

In one embodiment, the method is for inhibiting gene expression in amacrophage, a fibroblast, or a liver cell. In another embodiment, themethod is for activating gene expression in a macrophage, a fibroblast,or a liver cell.

In another embodiment, the method is for inhibiting gene expression in aneuronal cell. In another embodiment, the method is for activating geneexpression in a neuronal cell.

In other aspects, the invention provides methods for treating,preventing, reversing, or managing pathological processes mediated byMylip/Idol expression, such as a lipid disorder. In one embodiment, themethod includes administering to a patient in need of such treatment,prevention, reversal, or management a therapeutically orprophylactically effective amount of one or more of the iRNAs featuredin the invention. In one embodiment the patient has diabetes oratherosclerosis. In another embodiment, administration of the iRNAtargeting Mylip/Idol alleviates or relieves the severity of at least onesymptom of a Mylip/Idol-mediated disorder in the patient, such as highLDLc level, high ApoB level, or high total cholesterol level. In anotherembodiment, administration of the Mylip/Idol dsRNA does not lower thelevel of HDL cholesterol in the patient. In another embodiment,administration of the Mylip/Idol dsRNA increases neurite outgrowthand/or reduces at least one symptom of a neurodegenerative disease.

In one aspect, the invention provides a vector for inhibiting theexpression of a Mylip/Idol gene in a cell. In one embodiment, the vectorincludes at least one regulatory sequence operably linked to anucleotide sequence that encodes at least one strand of an iRNA asdescribed herein.

In another aspect, the invention provides a cell containing a vector forinhibiting the expression of a Mylip/Idol gene in a cell. The vectorincludes a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the iRNAs as describedherein.

In yet another aspect, the invention provides a composition containing aMylip/Idol iRNA, in combination with a second iRNA targeting a secondgene involved in a pathological disease, and useful for treating thedisease, e.g., a lipid disorder, metabolic disorder or neurodegenerativedisorder.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is the sequence of human Mylip/Idol mRNA (Ref. Seq.NM_(—)013262.3, SEQ ID NO: 644).

FIG. 2 is a sequence of mouse Mylip/Idol mRNA, isoform 1 (Ref. Seq.NM_(—)153789.3; SEQ ID NO: 645).

FIG. 3 is a sequence of mouse Mylip/Idol mRNA, isoform 2 (Ref. Seq.NM_(—)181043.1; SEQ ID NO: 646).

FIG. 4 is a sequence of rat Mylip/Idol mRNA (Ref. Seq.NM_(—)001107344.1; SEQ ID NO: 647).

DETAILED DESCRIPTION OF THE INVENTION

Described herein are iRNAs and methods of using them for inhibiting theexpression of a Mylip/Idol gene in a cell or a mammal where the iRNAtargets a Mylip/Idol gene. Also provided are compositions and methodsfor treating pathological conditions and diseases, such as a lipiddisorder, neurodegenerative disease, or a metabolic disorder, in amammal caused by the expression of a Mylip/Idol gene. iRNA directs thesequence-specific degradation of mRNA through a process known as RNAinterference (RNAi). In one embodiment, the iRNA activates theexpression of a Mylip/Idol gene in a cell or mammal, where the iRNAtargets a Mylip/Idol gene.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

The iRNAs of the compositions described herein include an RNA strand(the antisense strand) having a region which is 30 nucleotides or lessin length, i.e., 15-30 nucleotides in length, generally 19-24nucleotides in length, which region is substantially complementary to atleast part of an mRNA transcript of a Mylip/Idol gene. The use of theseiRNAs enables the targeted degradation of mRNAs of genes that areimplicated in pathologies associated with Mylip/Idol expression inmammals. Very low dosages of Mylip/Idol iRNAs in particular canspecifically and efficiently mediate RNAi, resulting in significantinhibition of expression of a Mylip/Idol gene. Using cell-based assays,the present inventors have demonstrated that iRNAs targeting Mylip/Idolcan specifically and efficiently mediate RNAi, resulting in significantinhibition of expression of a Mylip/Idol gene. Thus, methods andcompositions including these iRNAs are useful for treating pathologicalprocesses that can be mediated by down regulating Mylip/Idol, such as inthe treatment of a lipid disorder, e.g., atherosclerosis, andhypercholesterolemia. The following detailed description discloses howto make and use compositions containing iRNAs to inhibit the expressionof a Mylip/Idol gene, as well as compositions and methods for treatingdiseases and disorders caused by the expression of this gene.

Embodiments of the pharmaceutical compositions featured herein alsoinclude an iRNA having an antisense strand comprising a region which is30 nucleotides or less in length, generally 19-24 nucleotides in length,which region is substantially complementary to at least part of an RNAtranscript of a Mylip/Idol gene, together with a pharmaceuticallyacceptable carrier. Embodiments of compositions featured in theinvention also include an iRNA having an antisense strand having aregion of complementarity which is 30 nucleotides or less in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an RNA transcript of a Mylip/Idolgene.

Accordingly, in some aspects, pharmaceutical compositions containing aMylip/Idol iRNA and a pharmaceutically acceptable carrier, methods ofusing the compositions to inhibit expression of a Mylip/Idol gene, andmethods of using the pharmaceutical compositions to treat diseasescaused by expression of a Mylip/Idol gene are featured in the invention.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured herein by a nucleotidecontaining, for example, inosine. In another example, adenine andcytosine anywhere in the oligonucleotide can be replaced with guanineand uracil, respectively to form G-U Wobble base pairing with the targetmRNA. Sequences containing such replacement moieties are suitable forthe compositions and methods described herein.

As used herein, “Myosin regulatory light chain interacting protein”(“Mylip”) or “inducible degrador of the LDL-R” (“Idol”) refers to aparticular polypeptide expressed in a cell. Mylip is also known as Idol,Mylip/Idol, MIR (myosin regulatory light chain (MRLC) interactingprotein) and MSAP. The sequence of a human Mylip/Idol mRNA transcriptcan be found at NM_(—)013262.3 (SEQ ID NO: 644). The sequence of mouseMylip/Idol mRNA can be found at NM_(—)153789.3 (isoform 1; SEQ ID NO:645) or NM_(—)181043.1 (isoform 2; SEQ ID NO: 646), and the sequence ofrat Mylip/Idol mRNA can be found at NM_(—)001107344.1 (SEQ ID NO: 647).The mouse and rat sequences of Mylip/Idol are highly conserved.

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein effects inhibition ofMylip/Idol expression. Alternatively, in another embodiment, an iRNA asdescribed herein activates Mylip/Idol expression.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a Mylip/Idol gene, including messenger RNA (mRNA) that is a productof RNA processing of a primary transcription product. The target portionof the sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion. For example, the targetsequence will generally be from 9-36 nucleotides in length, e.g., 15-30nucleotides in length, including all sub-ranges therebetween. Asnon-limiting examples, the target sequence can be from 15-30nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides,15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides,18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides,19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides,20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides,21-23 nucleotides, or 21-22 nucleotides.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as can beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs (bp), whileretaining the ability to hybridize under the conditions most relevant totheir ultimate application, e.g., inhibition of gene expression via aRISC pathway. However, where two oligonucleotides are designed to form,upon hybridization, one or more single stranded overhangs, suchoverhangs shall not be regarded as mismatches with regard to thedetermination of complementarity. For example, a dsRNA comprising oneoligonucleotide 21 nucleotides in length and another oligonucleotide 23nucleotides in length, wherein the longer oligonucleotide comprises asequence of 21 nucleotides that is fully complementary to the shorteroligonucleotide, may yet be referred to as “fully complementary” for thepurposes described herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (an mRNA) refers to a polynucleotidethat is substantially complementary to a contiguous portion of the mRNAof interest (e.g., an mRNA encoding Mylip/Idol). For example, apolynucleotide is complementary to at least a part of a Mylip/Idol mRNAif the sequence is substantially complementary to a non-interruptedportion of an mRNA encoding Mylip/Idol.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to aniRNA that includes an RNA molecule or complex of molecules having ahybridized duplex region that comprises two anti-parallel andsubstantially complementary nucleic acid strands, which will be referredto as having “sense” and “antisense” orientations with respect to atarget RNA. The duplex region can be of any length that permits specificdegradation of a desired target RNA through a RISC pathway, but willtypically range from 9 to 36 base pairs in length, e.g., 15-30 basepairs in length. Considering a duplex between 9 and 36 base pairs, theduplex can be any length in this range, for example, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, or 36 and any sub-range therein between, including, butnot limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs,15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs,15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs,18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs,19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs,19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs,20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs,20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs,21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAsgenerated in the cell by processing with Dicer and similar enzymes aregenerally in the range of 19-22 base pairs in length. One strand of theduplex region of a dsDNA comprises a sequence that is substantiallycomplementary to a region of a target RNA. The two strands forming theduplex structure can be from a single RNA molecule having at least oneself-complementary region, or can be formed from two or more separateRNA molecules. Where the duplex region is formed from two strands of asingle molecule, the molecule can have a duplex region separated by asingle stranded chain of nucleotides (herein referred to as a “hairpinloop”) between the 3′-end of one strand and the 5′-end of the respectiveother strand forming the duplex structure. The hairpin loop can compriseat least one unpaired nucleotide; in some embodiments the hairpin loopcan comprise at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 20, at least 23 or moreunpaired nucleotides. Where the two substantially complementary strandsof a dsRNA are comprised by separate RNA molecules, those molecules neednot, but can be covalently connected. Where the two strands areconnected covalently by means other than a hairpin loop, the connectingstructure is referred to as a “linker.” The term “siRNA” is also usedherein to refer to a dsRNA as described above.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature, but also analogs and derivatives of RNAcomprising one or more ribonucleotide/ribonucleoside analogs orderivatives as described herein or as known in the art. Strictlyspeaking, a “ribonucleoside” includes a nucleoside base and a ribosesugar, and a “ribonucleotide” is a ribonucleoside with one, two or threephosphate moieties. However, the terms “ribonucleoside” and“ribonucleotide” can be considered to be equivalent as used herein. TheRNA can be modified in the nucleobase structure or in theribose-phosphate backbone structure, e.g., as described herein below.However, the molecules comprising ribonucleoside analogs or derivativesmust retain the ability to form a duplex. As non-limiting examples, anRNA molecule can also include at least one modified ribonucleosideincluding but not limited to a 2′-O-methyl modified nucleoside, anucleoside comprising a 5′ phosphorothioate group, a terminal nucleosidelinked to a cholesteryl derivative or dodecanoic acid bisdecylamidegroup, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoromodified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modifiednucleoside, morpholino nucleoside, a phosphoramidate or a non-naturalbase comprising nucleoside, or any combination thereof. Alternatively,an RNA molecule can comprise at least two modified ribonucleosides, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20 or more, up to the entirelength of the dsRNA molecule. The modifications need not be the same foreach of such a plurality of modified ribonucleosides in an RNA molecule.In one embodiment, modified RNAs contemplated for use in methods andcompositions described herein are peptide nucleic acids (PNAs) that havethe ability to form the required duplex structure and that permit ormediate the specific degradation of a target RNA via a RISC pathway.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside.In such an instance, an iRNA agent can comprise one or moredeoxynucleosides, including, for example, a deoxynucleoside overhang(s),or one or more deoxynucleosides within the double stranded portion of adsRNA. However, it is self evident that under no circumstances is adouble stranded DNA molecule encompassed by the term “iRNA.”

In one aspect, an RNA interference agent includes a single stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, long double strandedRNA introduced into plants and invertebrate cells is broken down intosiRNA by a Type III endonuclease known as Dicer (Sharp et al., GenesDev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). ThesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling thecomplementary antisense strand to guide target recognition (Nykanen, etal., (2001) Cell 107:309). Upon binding to the appropriate target mRNA,one or more endonucleases within the RISC cleaves the target to inducesilencing (Elbashir, et al.,(2001) Genes Dev. 15:188). Thus, in oneaspect the invention relates to a single stranded RNA that promotes theformation of a RISC complex to effect silencing of the target gene.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) may be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′ end, 3′ end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotideoverhang at the 3′ end and/or the 5′ end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/orthe 5′ end. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that issubstantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches may be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, in one embodiment, the term “SNALP” refers to a stablenucleic acid-lipid particle. A SNALP represents a vesicle of lipidscoating a reduced aqueous interior comprising a nucleic acid such as aniRNA or a plasmid from which an iRNA is transcribed. SNALPs aredescribed, e.g., in U.S. Patent Application Publication Nos.20060240093, 20070135372, and in International Application No. WO2009082817. Examples of “SNALP” formulations are described elsewhereherein.

“Introducing into a cell,” when referring to an iRNA, means facilitatingor effecting uptake or absorption into the cell, as is understood bythose skilled in the art. Absorption or uptake of an iRNA can occurthrough unaided diffusive or active cellular processes, or by auxiliaryagents or devices. The meaning of this term is not limited to cells invitro; an iRNA can also be “introduced into a cell,” wherein the cell ispart of a living organism. In such an instance, introduction into thecell will include the delivery to the organism. For example, for in vivodelivery, iRNA can be injected into a tissue site or administeredsystemically. In vivo delivery can also be by a beta-glucan deliverysystem, such as those described in U.S. Pat. Nos. 5,032,401 and5,607,677, and U.S. Publication No. 2005/0281781 which are herebyincorporated by reference in their entirety. In vitro introduction intoa cell includes methods known in the art such as electroporation andlipofection. Further approaches are described herein below or are knownin the art.

As used herein, the term “modulate the expression of,” refers to at anleast partial “inhibition” or partial “activation” of Mylip/Idol geneexpression in a cell treated with an iRNA composition as describedherein compared to the expression of Mylip/Idol in an untreated cell.

The terms “activate,” “enhance,” “up-regulate the expression of,”“increase the expression of,” and the like, in so far as they refer to aMylip/Idol gene, herein refer to the at least partial activation of theexpression of a Mylip/Idol gene, as manifested by an increase in theamount of Mylip/Idol mRNA, which can be isolated from or detected in afirst cell or group of cells in which a Mylip/Idol gene is transcribedand which has or have been treated such that the expression of aMylip/Idol gene is increased, as compared to a second cell or group ofcells substantially identical to the first cell or group of cells butwhich has or have not been so treated (control cells).

In one embodiment, expression of a Mylip/Idol gene is activated by atleast about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% byadministration of an iRNA as described herein. In some embodiments, aMylip/Idol gene is activated by at least about 60%, 70%, or 80% byadministration of an iRNA featured in the invention. In someembodiments, expression of a Mylip/Idol gene is activated by at leastabout 85%, 90%, or 95% or more by administration of an iRNA as describedherein. In some embodiments, the Mylip/Idol gene expression is increasedby at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold,at least 50-fold, at least 100-fold, at least 500-fold, at least 1000fold or more in cells treated with an iRNA as described herein comparedto the expression in an untreated cell. Activation of expression bysmall dsRNAs is described, for example, in Li et al., 2006 Proc. Natl.Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848,each of which is incorporated herein by reference.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in so far asthey refer to a Mylip/Idol gene, herein refer to the at least partialsuppression of the expression of a Mylip/Idol gene, as manifested by areduction of the amount of Mylip/Idol mRNA which can be isolated from ordetected in a first cell or group of cells in which a Mylip/Idol gene istranscribed and which has or have been treated such that the expressionof a Mylip/Idol gene is inhibited, as compared to a second cell or groupof cells substantially identical to the first cell or group of cells butwhich has or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition can be given in terms of areduction of a parameter that is functionally linked to Mylip/Idol geneexpression, e.g., the amount of protein encoded by a Mylip/Idol gene, orthe number of cells displaying a certain phenotype, e.g., stabilizationof microtubules. In principle, Mylip/Idol gene silencing can bedetermined in any cell expressing Mylip/Idol, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given iRNA inhibitsthe expression of the Mylip/Idol gene by a certain degree and thereforeis encompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of a Mylip/Idol gene issuppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50% by administration of an iRNA featured in the invention. In someembodiments, a Mylip/Idol gene is suppressed by at least about 60%, 70%,or 80% by administration of an iRNA described herein. In someembodiments, a Mylip/Idol gene is suppressed by at least about 85%, 90%,95%, 98%, 99%, or more, by administration of an iRNA as describedherein.

As used herein in the context of Mylip/Idol expression, the terms“treat,” “treatment,” and the like, refer to relief from or alleviationof pathological processes mediated by Mylip/Idol expression. In thecontext of the present invention insofar as it relates to any of theother conditions recited herein below (other than pathological processesmediated by Mylip/Idol expression), the terms “treat,” “treatment,” andthe like mean to relieve or alleviate at least one symptom associatedwith such condition, or to slow or reverse the progression oranticipated progression of such condition, such as slowing theprogression of a lipid disorder, such as atherosclerosis.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by Mylip/Idol expression or an overtsymptom of pathological processes mediated by Mylip/Idol expression. Thespecific amount that is therapeutically effective can be readilydetermined by an ordinary medical practitioner, and can vary dependingon factors known in the art, such as, for example, the type ofpathological processes mediated by Mylip/Idol expression, the patient'shistory and age, the stage of pathological processes mediated byMylip/Idol expression, and the administration of other agents thatinhibit pathological processes mediated by Mylip/Idol expression.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of an iRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an iRNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 10% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a10% reduction in that parameter. For example, a therapeuticallyeffective amount of an iRNA targeting Mylip/Idol can reduce Mylip/Idolprotein levels by at least 10%.

As used herein, the term “neurodegenerative disease” refers to adisorder of the central nervous system including e.g.: intracerebralhemorrhage (ICH), neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease and other degenerative diseases of thebasal ganglia; other neurological causes of memory loss or impairment,including Down's syndrome, Creutzfeldt-Jakob disease, prion diseases,cerebral ischemia and stroke; multiple sclerosis; motor neuron disease,such as amyotropic lateral sclerosis; neurological viral disease;Huntington's disease; hereditary spastic hemiplegia; primary lateralsclerosis; spinal muscular atrophy; Kennedy's disease; Shy-Dragersyndrome; Progressive Supranuclear Palsy; Lewy Body Disease;neuronopathies; dementia; frontotemporal lobe dementia; affectivedisorders (e.g. stress, depression and post-traumatic depression);neuropsychiatric disorders (e.g. schizophrenia, multiple sclerosis, andepilepsy); learning and memory disorders; and ocular neuron disorders)trigeminal neuralgia; glossopharyngeal neuralgia; Bell's Palsy;myasthenia gravis; progressive muscular atrophy; progressive bulbarinherited muscular atrophy; herniated, cervical spondylosis; plexusdisorders; thoracic outlet destruction syndromes; peripheralneuropathies; prophyria; muscular dystrophy; a polyglutamine repeatdisease; and spongiform encephalopathy. In one embodiment, theneurodegenerative disease is a result of injury or trauma and includese.g., post-surgical neurological dysfunction; ischemic disorders (e.g.cerebral or spinal cord infarction and ischemia, chronic ischemic braindisease, and stroke); kaumas (e.g. caused by physical injury or surgery,and compression injuries); ruptured and prolapsed invertebrate disksyndromes, among others. Ocular neuron disorders can also be treatedwith the methods and compositions described herein and include, but arenot limited to, retina or optic nerve disorders; optic nerve damage andoptic neuropathies such as Lebers hereditary optic neuropathy, autosomaldominant optic atrophy, optic neuritis; disorders of the optic nerve orvisual pathways; toxic neuropathies and toxic retinopathies; opticatrophy; glaucoma; retinal degenerations such as retinitis pigmentosa,macular degeneration, diabetic retinopathy.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. Agents included in drug formulations aredescribed further herein below.

As used herein, a “subject” is a mammal, e.g. a dog, horse, cat, andother non-human primates. In a preferred embodiment, a subject is ahuman.

As used herein, the term “LNPXX”, wherein the “XX” are numerals, is alsoreferred to as “AFXX” herein. For example, LNP09 is also referred toAF09 and LNP12 is also known as or referred to as AF12.

As used herein, the term “comprising” or “comprises” is used inreference to compositions, methods, and respective component(s) thereof,that are essential to the invention, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein, the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

II. Double-stranded Ribonucleic Acid (dsRNA)

Described herein are iRNA agents that modulate the expression of theMylip/Idol gene. In one embodiment, the iRNA agent includesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of a Mylip/Idol gene in a cell or mammal, e.g., in a humanhaving a lipid disorder, where the dsRNA includes an antisense strandhaving a region of complementarity which is complementary to at least apart of an mRNA formed in the expression of a Mylip/Idol gene, and wherethe region of complementarity is 30 nucleotides or less in length,generally 19-24 nucleotides in length, and where the dsRNA, upon contactwith a cell expressing the Mylip/Idol gene, inhibits the expression ofthe Mylip/Idol gene by at least 10% as assayed by, for example, a PCR orbranched DNA (bDNA)-based method, or by a protein-based method, such asby Western blot. In one embodiment, the iRNA agent activates theexpression of a Mylip/Idol gene in a cell or mammal. Expression of aMylip/Idol gene in cell culture, such as in COS cells, HeLa cells,primary hepatocytes, HepG2 cells, primary cultured cells or in abiological sample from a subject, can be assayed by measuring Mylip/IdolmRNA levels, such as by bDNA or TaqMan assay, or by measuring proteinlevels, such as by immunofluorescence analysis, using, for example,Western blotting or flow cytometric techniques.

A dsRNA includes two RNA strands that are complementary to hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of aMylip/Idol gene. The other strand (the sense strand) includes a regionthat is complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30inclusive, more generally between 18 and 25 inclusive, yet moregenerally between 19 and 24 inclusive, and most generally between 19 and21 base pairs in length, inclusive. Similarly, the region ofcomplementarity to the target sequence is between 15 and 30 inclusive,more generally between 18 and 25 inclusive, yet more generally between19 and 24 inclusive, and most generally between 19 and 21 nucleotides inlength, inclusive. In some embodiments, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of 9 to 36,e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that itbecomes processed to a functional duplex of e.g., 15-30 base pairs thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, then, an miRNA is a dsRNA. In another embodiment, a dsRNA isnot a naturally occurring miRNA. In another embodiment, an iRNA agentuseful to target Mylip/Idol expression is not generated in the targetcell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs. The dsRNA can be synthesized bystandard methods known in the art as further discussed below, e.g., byuse of an automated DNA synthesizer, such as are commercially availablefrom, for example, Biosearch, Applied Biosystems, Inc. In oneembodiment, a Mylip/Idol gene is a human Mylip/Idol gene. In anotherembodiment the Mylip/Idol gene is a mouser or a rat Mylip/Idol gene. Inspecific embodiments, the first sequence is a sense strand of a dsRNAthat includes a sense sequence of one of Tables 3 and 5, and the secondsequence is selected from the group consisting of the antisensesequences of one of Tables 3 and 5. Alternative dsRNA agents that targetelsewhere in the target sequence provided in Tables 3 and 5 can readilybe determined using the target sequence and the flanking Mylip/Idolsequence.

In one aspect, a dsRNA will include at least two nucleotide sequences, asense and an antisense sequence, whereby the sense strand is selectedfrom the groups of sequences provided in Table 3 (SEQ ID NO: 20-SEQ IDNO: 167; SEQ ID NO: 648-SEQ ID NO: 1103), Table 4 (SEQ ID NO: 168-SEQ IDNO: 299), Table 5 (SEQ ID NO: 300-SEQ ID NO: 447), and Table 6 (SEQ IDNO: 448-SEQ ID NO: 579), and the corresponding antisense strand of thesense strand selected from Table 3 (SEQ ID NO: 20-SEQ ID NO: 167; SEQ IDNO: 648-SEQ ID NO: 1103), Table 4 (SEQ ID NO: 168-SEQ ID NO: 299), Table5 (SEQ ID NO: 300-SEQ ID NO: 447), and Table 6 (SEQ ID NO: 448-SEQ IDNO: 579). In this aspect, one of the two sequences is complementary tothe other of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of a Mylip/Idol gene. As such, in this aspect, a dsRNA willinclude two oligonucleotides, where one oligonucleotide is described asthe sense strand in Tables 3, 4, 5 and 6, and the second oligonucleotideis described as the corresponding antisense strand of the sense strandfrom Tables 3, 4, 5 and 6. As described elsewhere herein and as known inthe art, the complementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 3, 4, 5 and 6, dsRNAsdescribed herein can include at least one strand of a length ofminimally 21 nt. It can be reasonably expected that shorter duplexeshaving one of the sequences of Tables 3, 4, 5 and 6 minus only a fewnucleotides on one or both ends may be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a partial sequenceof at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides fromone of the sequences of Tables 3, 4, 5 and 6, and differing in theirability to inhibit the expression of a Mylip/Idol gene by not more than5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the fullsequence, are contemplated according to the invention.

In addition, the RNAs provided in Tables 3, 4, 5 and 6 identify a sitein a Mylip/Idol transcript that is susceptible to RISC-mediatedcleavage. As such, the present invention further features iRNAs thattarget within one of such sequences. As used herein, an iRNA is said totarget within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least 15 contiguousnucleotides from one of the sequences provided in Tables 3, 4, 5 and 6coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in a Mylip/Idol gene.

While a target sequence is generally 15-30 nucleotides in length, thereis wide variation in the suitability of particular sequences in thisrange for directing cleavage of any given target RNA. Various softwarepackages and the guidelines set out herein provide guidance for theidentification of optimal target sequences for any given gene target,but an empirical approach can also be taken in which a “window” or“mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that mayserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in Tables 3, 4, 5 and 6represent effective target sequences, it is contemplated that furtheroptimization of inhibition efficiency can be achieved by progressively“walking the window” one nucleotide upstream or downstream of the givensequences to identify sequences with equal or better inhibitioncharacteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 3, 4, 5 and 6, further optimization could be achieved bysystematically either adding or removing nucleotides to generate longeror shorter sequences and testing those and sequences generated bywalking a window of the longer or shorter size up or down the target RNAfrom that point. Again, coupling this approach to generating newcandidate targets with testing for effectiveness of iRNAs based on thosetarget sequences in an inhibition assay as known in the art or asdescribed herein can lead to further improvements in the efficiency ofinhibition. Further still, such optimized sequences can be adjusted by,e.g., the introduction of modified nucleotides as described herein or asknown in the art, addition or changes in overhang, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes, etc.) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch not be located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent RNA strandwhich is complementary to a region of a Mylip/Idol gene, the RNA strandgenerally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether an iRNA containing a mismatch to atarget sequence is effective in inhibiting the expression of aMylip/Idol gene. Consideration of the efficacy of iRNAs with mismatchesin inhibiting expression of a Mylip/Idol gene is important, especiallyif the particular region of complementarity in a Mylip/Idol gene isknown to have polymorphic sequence variation within the population.

In one embodiment, at least one end of a dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. Such dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties relative to their blunt-ended counterparts. In yetanother embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemicallymodified to enhance stability or other beneficial characteristics. Thenucleic acids featured in the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,(a) end modifications, e.g., 5′ end modifications (phosphorylation,conjugation, inverted linkages, etc.) 3′ end modifications (conjugation,DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases, (c) sugar modifications(e.g., at the 2′ position or 4′ position) or replacement of the sugar,as well as (d) backbone modifications, including modification orreplacement of the phosphodiester linkages. Specific examples of RNAcompounds useful in the embodiments described herein include, but arenot limited to, RNAs containing modified backbones or no naturalinternucleoside linkages. RNAs having modified backbones include, amongothers, those that do not have a phosphorus atom in the backbone. Forthe purposes of this specification, and as sometimes referenced in theart, modified RNAs that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.In particular embodiments, the modified RNA will have a phosphorus atomin its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, each of which is herein incorporated by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found, for example,in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, each of which is herein incorporated byreference in its entirety.

Another modification of the RNA of an iRNA featured in the inventioninvolves chemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution,pharmacokinetic properties, or cellular uptake of the iRNA. Suchmoieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989,86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994, 4:1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a PK modulator. As used herein, a “PK modulator” refers to apharmacokinetic modulator. PK modulators include lipophiles, bile acids,steroids, phospholipid analogues, peptides, protein binding agents, PEG,vitamins etc. Examplary PK modulators include, but are not limited to,cholesterol, fatty acids, cholic acid, lithocholic acid,dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides thatcomprise a number of phosphorothioate linkages are also known to bind toserum protein, thus short oligonucleotides, e.g., oligonucleotides ofabout 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple ofphosphorothioate linkages in the backbaone are also amenable to thepresent invention as ligands (e.g. as PK modulating ligands). Inaddition, aptamers that bind serum components (e.g. serum proteins) arealso suitable for use as PK modulating ligands in the embodimentsdescribed herein.

For macromolecular drugs and hydrophilic drug molecules, which cannoteasily cross bilayer membranes, entrapment in endosomal/lysosomalcompartments of the cell is thought to be the biggest hurdle foreffective delivery to their site of action. In recent years, a number ofapproaches and strategies have been devised to address this problem. Forliposomal formulations, the use of fusogenic lipids in the formulationhave been the most common approach (Singh, R. S., Goncalves, C. et al.(2004). On the Gene Delivery Efficacies of pH-Sensitive Cationic Lipidsvia Endosomal Protonation. A Chemical Biology Investigation. Chem. Biol.11, 713-723.). Other components, which exhibit pH-sensitiveendosomolytic activity through protonation and/or pH-inducedconformational changes, include charged polymers and peptides. Examplesmay be found in Hoffman, A. S., Stayton, P. S. et al. (2002). Design of“smart” polymers that can direct intracellular drug delivery. PolymersAdv. Technol. 13, 992-999; Kakudo, Chaki, T., S. et al. (2004).Transferrin-Modified Liposomes Equipped with a pH-Sensitive FusogenicPeptide: An Artificial Viral-like Delivery System. Biochemistry 436,5618-5628; Yessine, M. A. and Leroux, J. C. (2004).Membrane-destabilizing polyanions: interaction with lipid bilayers andendosomal escape of biomacromolecules. Adv. Drug Deliv. Rev. 56,999-1021; Oliveira, S., van Rooy, I. et al. (2007). Fusogenic peptidesenhance endosomal escape improving iRNA-induced silencing of oncogenes.Int. J. Pharm. 331, 211-4. They have generally been used in the contextof drug delivery systems, such as liposomes or lipoplexes. For folatereceptor-mediated delivery using liposomal formulations, for instance, apH-sensitive fusogenic peptide has been incorporated into the liposomesand shown to enhance the activity through improving the unloading ofdrug during the uptake process (Turk, M. J., Reddy, J. A. et al. (2002).Characterization of a novel pH-sensitive peptide that enhances drugrelease from folate-targeted liposomes at endosomal pHs is described inBiochim. Biophys. Acta 1559, 56-68).

In certain embodiments, the endosomolytic components of the presentinvention can be polyanionic peptides or peptidomimetics which showpH-dependent membrane activity and/or fusogenicity. A peptidomimetic canbe a small protein-like chain designed to mimic a peptide. Apeptidomimetic can arise from modification of an existing peptide inorder to alter the molecule's properties, or the synthesis of apeptide-like molecule using unnatural amino acids or their analogs. Incertain embodiments, they have improved stability and/or biologicalactivity when compared to a peptide. In certain embodiments, theendosomolytic component assumes its active conformation at endosomal pH(e.g., pH 5-6). The “active” conformation is that conformation in whichthe endosomolytic component promotes lysis of the endosome and/ortransport of the modular composition of the invention, or its any of itscomponents (e.g., a nucleic acid), from the endosome to the cytoplasm ofthe cell.

Libraries of compounds can be screened for their differential membraneactivity at endosomal pH versus neutral pH using a hemolysis assay.Promising candidates isolated by this method may be used as componentsof the modular compositions of the invention. A method for identifyingan endosomolytic component for use in the compositions and methods ofthe present invention may comprise: providing a library of compounds;contacting blood cells with the members of the library, wherein the pHof the medium in which the contact occurs is controlled; determiningwhether the compounds induce differential lysis of blood cells at a lowpH (e.g., about pH 5-6) versus neutral pH (e.g., about pH 7-8).

Exemplary endosomolytic components include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component can contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of endosomolytic componentsinclude H2N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO2H (SEQ ID NO: 1104);H2N-(AALAEALAEALAEALAEALAEALAAAAGGC)-CO2H (SEQ ID NO: 1105); andH2N-(ALEALAEALEALAEA)-CONH2 (SEQ ID NO: 1106).

In certain embodiments, more than one endosomolytic component can beincorporated into the iRNA agent of the invention. In some embodiments,this will entail incorporating more than one of the same endosomolyticcomponent into the iRNA agent. In other embodiments, this will entailincorporating two or more different endosomolytic components into iRNAagent.

These endosomolytic components can mediate endosomal escape by, forexample, changing conformation at endosomal pH. In certain embodiments,the endosomolytic components can exist in a random coil conformation atneutral pH and rearrange to an amphipathic helix at endosomal pH. As aconsequence of this conformational transition, these peptides may insertinto the lipid membrane of the endosome, causing leakage of theendosomal contents into the cytoplasm. Because the conformationaltransition is pH-dependent, the endosomolytic components can displaylittle or no fusogenic activity while circulating in the blood (pH˜7.4).“Fusogenic activity,” as used herein, is defined as that activity whichresults in disruption of a lipid membrane by the endosomolyticcomponent. One example of fusogenic activity is the disruption of theendosomal membrane by the endosomolytic component, leading to endosomallysis or leakage and transport of one or more components of the modularcomposition of the invention (e.g., the nucleic acid) from the endosomeinto the cytoplasm.

In addition to hemolysis assays, as described herein, suitableendosomolytic components can be tested and identified by a skilledartisan using other methods. For example, the ability of a compound torespond to, e.g., change charge depending on, the pH environment can betested by routine methods, e.g., in a cellular assay. In certainembodiments, a test compound is combined with or contacted with a cell,and the cell is allowed to internalize the test compound, e.g., byendocytosis. An endosome preparation can then be made from the contactedcells and the endosome preparation compared to an endosome preparationfrom control cells. A change, e.g., a decrease, in the endosome fractionfrom the contacted cell vs. the control cell indicates that the testcompound can function as a fusogenic agent. Alternatively, the contactedcell and control cell can be evaluated, e.g., by microscopy, e.g., bylight or electron microscopy, to determine a difference in the endosomepopulation in the cells. The test compound and/or the endosomes canlabeled, e.g., to quantify endosomal leakage.

In another type of assay, an iRNA agent described herein is constructedusing one or more test or putative fusogenic agents. The iRNA agent canbe labeled for easy visulization. The ability of the endosomolyticcomponent to promote endosomal escape, once the iRNA agnet is taken upby the cell, can be evaluated, e.g., by preparation of an endosomepreparation, or by microscopy techniques, which enable visualization ofthe labeled iRNA agent in the cytoplasm of the cell. In certain otherembodiments, the inhibition of gene expression, or any otherphysiological parameter, may be used as a surrogate marker for endosomalescape.

In other embodiments, circular dichroism spectroscopy can be used toidentify compounds that exhibit a pH-dependent structural transition.

A two-step assay can also be performed, wherein a first assay evaluatesthe ability of a test compound alone to respond to changes in pH, and asecond assay evaluates the ability of a modular composition thatincludes the test compound to respond to changes in pH.

Lipid Conjugates

In one ligand, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

Cell Permeation Peptides

Peptides suitable for use with the present invention can be a naturalpeptide, .e.g., tat or antennopedia peptide, a synthetic peptide, or apeptidomimetic. Furthermore, the peptide can be a modified peptide, forexample peptide can comprise non-peptide or pseudo-peptide linkages, andD-amino acids. A peptidomimetic (also referred to herein as anoligopeptidomimetic) is a molecule capable of folding into a definedthree-dimensional structure similar to a natural peptide. The attachmentof peptide and peptidomimetics to iRNA agents can affect pharmacokineticdistribution of the iRNA, such as by enhancing cellular recognition andabsorption. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:16). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:17)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:18)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:19))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably, the peptide or peptidomimetic tethered tothe lipid is a cell-targeting peptide such as anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an dsRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver a iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an αvβ3 integrin. Thus,one could use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the αvβ3 integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogeneis.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

Carbohydrate Conjugates

In some embodiments, the iRNA oligonucleotides described herein furthercomprise carbohydrate conjugates. The carbohydrate conjugates areadvantageous for the in vivo delivery of nucleic acids, as well ascompositions suitable for in vivo therapeutic use, as described herein.As used herein, “carbohydrate” refers to a compound which is either acarbohydrate per se made up of one or more monosaccharide units havingat least 6 carbon atoms (which may be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which may be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri- and oligosaccharides containingfrom about 4-9 monosaccharide units), and polysaccharides such asstarches, glycogen, cellulose and polysaccharide gums. Specificmonosaccharides include C₅ and above (preferably C₅-C₈) sugars; di- andtrisaccharides include sugars having two or three monosaccharide units(preferably C₅-C₈).

In one embodiment, the carbohydrate conjugate is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises otherligand such as, but not limited to, PK modulator, endosomolytic ligand,and cell permeation peptide.

Linkers

In some embodiments, the conjugates described herein can be attached tothe iRNA oligonucleotide with various linkers that can be cleavable ornon cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound. Linkers typically comprise a directbond or an atom such as oxygen or sulfur, a unit such as NR⁸, C(O),C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl,arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R⁸), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R⁸ is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between 1-24atoms, preferably 4-24 atoms, preferably 6-18 atoms, more preferably8-18 atoms, and most preferably 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)— (SEQ ID NO: 1107), where R^(A) and R^(B)are the R groups of the two adjacent amino acids. These candidates canbe evaluated using methods analogous to those described above.

Representative carbohydrate conjugates with linkers include, but are notlimited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or“chimeras,” in the context of this invention, are iRNA compounds,preferably dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These iRNAs typically contain at leastone region wherein the RNA is modified so as to confer upon the iRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the iRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of iRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter iRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxy dsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved ina number of different ways. In vivo delivery can be performed directlyby administering a composition comprising an iRNA, e.g. a dsRNA, to asubject. Alternatively, delivery can be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA.

Direct Delivery of an iRNA Composition

In general, any method of delivering a nucleic acid molecule can beadapted for use with an iRNA (see e.g., Akhtar S, and Julian R L. (1992)Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporatedherein by reference in their entireties). However, there are threefactors that are important to consider in order to successfully deliveran iRNA molecule in vivo: (a) biological stability of the deliveredmolecule, (2) preventing non-specific effects, and (3) accumulation ofthe delivered molecule in the target tissue. The non-specific effects ofan iRNA can be minimized by local administration, for example by directinjection or implantation into a tissue (as a non-limiting example, atumor) or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that may otherwise beharmed by the agent or that may degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, DR., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

Vector Encoded dsRNAs

In another aspect, iRNA targeting the Mylip/Idol gene can be expressedfrom transcription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, PCT PublicationNo. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct maybe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an iRNA can be used. For example, a retroviral vectorcan be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)).These retroviral vectors contain the components necessary for thecorrect packaging of the viral genome and integration into the host cellDNA. The nucleic acid sequences encoding an iRNA are cloned into one ormore vectors, which facilitates delivery of the nucleic acid into apatient. More detail about retroviral vectors can be found, for example,in Boesen et al., Biotherapy 6:291-302 (1994), which describes the useof a retroviral vector to deliver the mdr1 gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993). Lentiviral vectors contemplated for useinclude, for example, the HIV based vectors described in U.S. Pat. Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated byreference.

Adenoviruses are also contemplated for use in delivery of iRNAs.Adenoviruses are especially attractive vehicles, e.g., for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walshet al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). In one embodiment, the iRNA can be expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector having, for example, either the U6 or H1 RNA promoters, orthe cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressingthe dsRNA featured in the invention, methods for constructing therecombinant AV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S.Pat. No. 5,139,941; International Patent Application No. WO 94/13788;and International Patent Application No. WO 93/24641, the entiredisclosures of which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus,for example an attenuated vaccinia such as Modified Virus Ankara (MVA)or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

III. Pharmaceutical Compositions Containing Irna

In one embodiment, provided herein are pharmaceutical compositionscontaining an iRNA and a pharmaceutically acceptable carrier. Thepharmaceutical composition containing the iRNA is useful for treating adisease or disorder associated with the expression or activity of aMylip/Idol gene, such as pathological processes mediated by Mylip/Idolexpression. Such pharmaceutical compositions are formulated based on themode of delivery. One example is compositions that are formulated forsystemic administration via parenteral delivery, e.g., by intravenous(IV) delivery. Another example is compositions that are formulated fordirect delivery into the brain parenchyma, e.g., by infusion into thebrain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of Mylip/Idol genes. Ingeneral, a suitable dose of iRNA will be in the range of 0.01 to 200.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 to 50 mg per kilogram body weight per day. Forexample, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition maybe administered once daily, or the iRNA may be administered as two,three, or more sub-doses at appropriate intervals throughout the day oreven using continuous infusion or delivery through a controlled releaseformulation. In that case, the iRNA contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation which providessustained release of the iRNA over a several day period. Sustainedrelease formulations are well known in the art and are particularlyuseful for delivery of agents at a particular site, such as could beused with the agents of the present invention. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on Mylip/Idol levels can be long lasting,such that subsequent doses are administered at not more than 3, 4, or 5day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by Mylip/Idol expression. Such models can be used for in vivotesting of iRNA, as well as for determining a therapeutically effectivedose. A suitable mouse model is, for example, a mouse containing atransgene expressing human Mylip/Idol.

The present invention also includes pharmaceutical compositions andformulations that include the iRNA compounds featured in the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver nucleicacids encoding the thymidine kinase gene to cell monolayers in culture.Expression of the exogenous gene was detected in the target cells (Zhouet al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describes PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, an Idol/Mylip dsRNA featured in the invention isfully encapsulated in the lipid formulation, e.g., to form a SPLP,pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, theterm “SNALP” refers to a stable nucleic acid-lipid particle, includingSPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipidparticle comprising plasmid DNA encapsulated within a lipid vesicle.SNALPs and SPLPs typically contain a cationic lipid, a non-cationiclipid, and a lipid that prevents aggregation of the particle (e.g., aPEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 nm toabout 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid may comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40%2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which is hereinincorporated by reference in its entirety), Cholesterol (Sigma-Aldrich),and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are as follows:

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugateCationic Lipid Lipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N-DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA)(57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 S-XTC2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/CholesteroyPEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-C12-200/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:11-yl)ethylazanediyl)didodecan-2-ol (C12-200) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference. XTC comprising formulations are described, e.g., in U.S.Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, which is herebyincorporated by reference. MC3 comprising formulations are described,e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S.Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and InternationalApplication No. PCT/US10/28224, filed Jun. 10, 2010, which are herebyincorporated by reference. ALNY-100 comprising formulations aredescribed, e.g., International patent application number PCT/US09/63933,filed on Nov. 10, 2009, which is hereby incorporated by reference.C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x),—C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y),wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different andindependently hydrogen, alkyl or heterocycle, and each of said alkyl andheterocycle substituents may be further substituted with one or more ofoxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments, an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above may be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R₃ and R₄ are independentlylower alkyl or R₃ and R₄ can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25(m,2H). LC-MS [M+H]-232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93(m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3,[M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27(m, 1H),5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m,1H), 4.58-4.57(m,2H),2.78-2.74(m,7H), 2.06-2.00(m, 8H), 1.96-1.91(m, 2H), 1.62(m,4H),1.48(m,2H), 1.37-1.25(br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR □=130.2, 130.1 (x2),127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7,29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+Calc. 654.6,Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants may beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of iRNAs through the mucosa is enhanced.In addition to bile salts and fatty acids, these penetration enhancersinclude, for example, sodium lauryl sulfate, polyoxyethylene-9-laurylether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92); and perfluorochemical emulsions, such asFC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of iRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. etal., Excipient development for pharmaceutical, biotechnology, and drugdelivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al.,J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of iRNAs throughthe alimentary mucosa (see e.g., Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33). This class ofpenetration enhancers includes, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellffectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreanti-cytokine biologic agents which function by a non-RNAi mechanism.Examples of such biologics include, biologics that target IL1β (e.g.,anakinra), IL6 (e.g., tocilizumab), or TNF (e.g., etanercept,infliximab, adlimumab, or certolizumab).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsdescribed herein can be administered in combination with other knownagents effective in treatment of pathological processes mediated byMylip/Idol expression. In any event, the administering physician canadjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

Methods for Treating Diseases Caused by Expression of a Mylip/Idol Gene

The invention relates in particular to the use of an iRNA targetingMylip/Idol and compositions containing at least one such iRNA for thetreatment of a Mylip/Idol-mediated disorder or disease. For example, acomposition containing an iRNA targeting a Mylip/Idol gene is used fortreatment of lipid or metabolic disorders, such as hypercholesterolemia,dyslipidemia, diabetes, diabetes type I, diabetes type II, coronaryartery disease, atherosclerosis, myocardial infarction, coronary arterybypass graft, percutaneous transluminal angioplasties, coronarystenosis, cerebrovascular disease transient ischemic attack, ischemicstroke, carotid endarterectomies, peripheral arterial disease, and otherdisorders associated with cholesterol metabolism.

The invention further relates to the use of an iRNA or a pharmaceuticalcomposition thereof, e.g., for treating a lipid disorder, in combinationwith other pharmaceuticals and/or other therapeutic methods, e.g., withknown pharmaceuticals and/or known therapeutic methods, such as, forexample, those which are currently employed for treating thesedisorders. For example, in certain embodiments, an iRNA targetingMylip/Idol is administered in combination with, e.g., an HMG-CoAreductase inhibitor (e.g., a statin, such as atrovastatin, lovastatin,pravastatin or simvastatin), a fibrate, a bile acid sequestrant, niacin,an antiplatelet agent, an angiotensin converting enzyme inhibitor, anangiotensin II receptor antagonist (e.g., losartan potassium, such asMerck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT)inhibitor, a cholesterol absorption inhibitor, a cholesterol estertransfer protein (CETP) inhibitor, a microsomal triglyceride transferprotein (MTTP) inhibitor, a cholesterol modulator, a bile acidmodulator, a peroxisome proliferation activated receptor (PPAR) agonist,a gene-based therapy, a composite vascular protectant (e.g., AGI-1067,from Atherogenics), a glycoprotein IIb/IIIa inhibitor, aspirin or anaspirin-like compound, an IBAT inhibitor (e.g., S-8921, from Shionogi),a squalene synthase inhibitor, or a monocyte chemoattractant protein(MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors includeatorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl),pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo'sMevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, BoehringerIngelheim's Denan, Banyu's Lipovas), lovastatin (Merck'sMevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma'sLiposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa'sCranoc, Solvay's Digaril), cerivastatin (Bayer'sLipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca'sCrestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical,Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g.,bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol),clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier'sLipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics),gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate(Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrantsinclude, e.g., cholestyramine (Bristol-Myers Squibb's Questran® andQuestran Light™), colestipol (e.g., Pharmacia's Colestid), andcolesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapiesinclude, e.g., immediate release formulations, such as Aventis' Nicobid,Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit.Niacin extended release formulations include, e.g., Kos Pharmaceuticals'Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agentsinclude, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel(Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine(e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Otheraspirin-like compounds useful in combination with an iRNA targetingMylip/Idol include, e.g., Asacard (slow-release aspirin, by Pharmacia)and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplaryangiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g.,Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplaryacyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g.,avasimibe (Pfizer), eflucimibe (BioM{acute over (ε)}rieux PierreFabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito).Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe(Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer).Exemplary CETP inhibitors include, e.g., Torcetrapib (also calledCP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (AvantImmunotherapeutics). Exemplary microsomal triglyceride transfer protein(MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757(Janssen), and CP-346086 (Pfizer). Other exemplary cholesterolmodulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027(Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulatorsinclude, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British TechnologyGroup), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), andAZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activatedreceptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242)(AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson),GW-409544 (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 (LigandPharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and EliLilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674(Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin).Exemplary gene-based therapies include, e.g., AdGWEGF121.10 (GenVec),ApoAl (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics),and ATP-binding cassette transporter-Al (ABCAl) (CV Therapeutics/Incyte,Aventis, Xenon). Exemplary Glycoprotein IIb/IIIa inhibitors include,e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban(Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).Exemplary squalene synthase inhibitors include, e.g., BMS-1884941(Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer),CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitoris, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agentBO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivativeNyclin (Yamanouchi Pharmaceuticals) are also appropriate foradministering in combination with an iRNA featured in the invention.Exemplary combination therapies suitable for administration with an iRNAtargeting Mylip/Idol include, e.g., advicor (Niacin/lovastatin from KosPharmaceuticals), amlodipine/atorvastatin (Pfizer), andezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treatinghypercholesterolemia, and suitable for administration in combinationwith an iRNA targeting Mylip/Idol include, e.g., lovastatin, niacinAltoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet®Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor®Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis),fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodiumLipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules(Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets(Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott),fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-PloughPharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo),colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia®Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor®Tablets (Merck).

In one embodiment, a dsRNA targeting Mylip/Idol is administered incombination with an ezetimibe/simvastatin combination (e.g., Vytorin®(Merck/Schering-Plough Pharmaceuticals)).

The invention further relates to the use of a dsRNA or a pharmaceuticalcomposition containing a dsRNA for treatment of a metabolic disorder,such as diabetes, in combination with other pharmaceuticals and/or othertherapeutic methods, e.g., with known pharmaceuticals and/or knowntherapeutic methods, such as, for example, those which are currentlyemployed for treating metabolic disorders (e.g., diabetes). For example,in certain embodiments, administration of a dsRNA targeting Mylip/Idolis administered in combination with, e.g., insulin (e.g., insulininjections); a biguanide (e.g., metformin); a sulfonylurea (e.g.,glibenclamide, glipizide, tolbautamide, chloropamidem, tolazamide,glimepride, glicazide or glyburide); an alpha-glucosidase inhibitor(e.g., acarbose); a PPAR gamma agonist (e.g., thiazolidinedione andderivatives such as rosiglitazone or pioglitazone); anoxadiazolidinedione; a meglitinide; a D-phenylalanine derivative;repaglinide; a PPAR (Peroxisome proliferator-activated receptor) ligandincluding the PPAR-alpha, PPAR-gamma and PPAR-delta subtypes; an RXR(retinoid X receptor) agonist, such as ALRT-268, LG-1268 or LG-1069; aPPAR alpha agonist (e.g., clofibrate and gemfibrozil); an alpha agonist(non-thiazolinedione); a glycogen phosphorylase inhibitor; aglucagon-like peptide; a dipeptidylpeptidase IV inhibitor; an HMG-CoAreductase inhibitor (e.g., a statin, such as atrovastatin, lovastatin,pravastatin or simvastatin); a GLP-1 antagonist; a DPP-IV (dipeptidylpeptidase-IV) inhibitor; a PTPase (protein tyrosine phosphatase)inhibitor; or a compound lowering food intake.

The iRNA and an additional therapeutic agent can be administered in thesame combination, e.g., parenterally, or the additional therapeuticagent can be administered as part of a separate composition or byanother method described herein.

The invention features a method of administering an iRNA agent targetingMylip/Idol to a patient having a disease or disorder mediated byMylip/Idol expression, such as a lipid disorder, or a disorderassociated with cholesterol metabolism, e.g., diabetes oratherosclerosis. Administration of the dsRNA can lower LDL levels, lowerApoB levels, or lower total cholesterol level, for example, in a patientwith a lipid disorder, or a disorder associated with cholesterolmetabolism. By “lower” in this context is meant a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 10%, at least 20%, at least 30%, at least 40% or more, and ispreferably down to a level accepted as within the range of normal for anindividual without such disorder.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, the levels of LDL cholesterol can be monitored for efficacy ofa given treatment regime. The iRNA treatments described herein can beused to treat individuals having moderately elevated plasma LDLcholesterol levels (e.g., 130-159 mg/dL), high LDL plasma levels (e.g.,160-189 mg/dL), or very high LDL cholesterol levels (e.g., 190 mg/dL).In addition, the treatment described herein may also be used to preventhigh LDL cholesterol levels in individuals with only minor elevations inLDL cholesterol (e.g., 100-129 mg/dL). One of skill in the art caneasily monitor the LDL levels in subjects receiving treatment with iRNAas described herein and assay for a reduction in LDL cholesterol levelsof at least 10% and preferably to a clinical level representing a lowrisk of LDL cholesterol mediated disease e.g., <100 mg/dL.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the NYHA Classes of Heart failure. In this example, thereare four stages of heart failure graded from mild to severe, based onsymptoms such as e.g., the ability to carry on physical activity,shortness of breath, and palpitations. Efficacy can be measured in thisexample by the movement of a patient from e.g., a Class IV (severe)heart failure profile to a Class III, Class II, or Class I heart failureprofile. Similar grading scales exist for many diseases and disorders,including, but not limited to heart disease, diabetic retinopathy,systemic sclerosis, Clostridium difficile-Associated Disease,Lipodystrophy (the Lipodystrophy Severity Grading Scale), HIV (the HIVOutpatient Study scale), cancer grading, cancer staging, etc., and canbe used to determine a patient's progress in response to treatment. Anypositive change resulting in e.g., lessening of severity of diseasemeasured using the appropriate scale, represents adequate treatmentusing an iRNA or iRNA formulation as described herein.

Patients can be administered a therapeutic amount of iRNA, such as 0.01mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0mg/kg, or 2.5 mg/kg dsRNA. The iRNA can be administered by intravenousinfusion over a period of time, such as over a 5 minute, 10 minute, 15minute, 20 minute, or 25 minute period. The administration is repeated,for example, on a regular basis, such as biweekly (i.e., every twoweeks) for one month, two months, three months, four months or longer.After an initial treatment regimen, the treatments can be administeredon a less frequent basis. For example, after administration biweekly forthree months, administration can be repeated once per month, for sixmonths or a year or longer. Administration of the iRNA can reduceMylip/Idol levels, e.g., in a cell, tissue, blood, urine or othercompartment of the patient by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction, or forelevated lipid levels or blood pressure. In another example, the patientcan be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Many lipid diseases and disorders are hereditary. Therefore, a patientin need of a Mylip/Idol iRNA may be identified by taking a familyhistory. A healthcare provider, such as a doctor, nurse, or familymember, can take a family history before prescribing or administering aMylip/Idol dsRNA. A DNA test may also be performed on the patient toidentify a mutation in the Mylip/Idol gene, before a Mylip/Idol dsRNA isadministered to the patient.

Owing to the inhibitory effects on Mylip/Idol expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

Methods for Modulating Expression of a Mylip/Idol Gene

In yet another aspect, the invention provides a method for modulating(e.g., inhibiting or activating) the expression of a Mylip/Idol gene ina mammal.

In one embodiment, the method includes administering a compositionfeatured in the invention to the mammal such that expression of thetarget Mylip/Idol gene is decreased, such as for an extended duration,e.g., at least two, three, four days or more, e.g., one week, two weeks,three weeks, or four weeks or longer. The effect of the decreased targetMylip/Idol gene preferably results in a decrease in LDLc levels in theblood, and more particularly in the serum, of the mammal. In someembodiments, LDLc levels are decreased by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, or atleast 60%, or more, as compared to pretreatment levels.

In another embodiment, the method includes administering a compositionas described herein to a mammal such that expression of the targetMylip/Idol gene is increased by e.g., at least 10% compared to anuntreated animal. In some embodiments, the activation of Mylip/Idoloccurs over an extended duration, e.g., at least two, three, four daysor more, e.g., one week, two weeks, three weeks, four weeks, or more.Without wishing to be bound by theory, an iRNA can activate Mylip/Idolexpression by stabilizing the Mylip/Idol mRNA transcript, interactingwith a promoter in the genome, and/or inhibiting an inhibitor ofMylip/Idol expression.

Preferably, the iRNAs useful for the methods and compositions featuredin the invention specifically target RNAs (primary or processed) of thetarget Mylip/Idol gene. Compositions and methods for inhibiting theexpression of these Mylip/Idol genes using iRNAs can be prepared andperformed as described elsewhere herein.

In one embodiment, the method includes administering a compositioncontaining an iRNA, where the iRNA includes a nucleotide sequence thatis complementary to at least a part of an RNA transcript of theMylip/Idol gene of the mammal to be treated. When the organism to betreated is a mammal such as a human, the composition may be administeredby any means known in the art including, but not limited to oral,intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1 Interference RNA (iRNA) Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Oligonucleotide Synthesis.

Applicants have used several different methods to generate the iRNAmolecules described herein. This Example describes one approach that hasbeen used. The ordinarily skilled artisan can use any method known inthe art to prepare iRNAs as described herein.

Oligonucleotides are synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG,500Å, Prime Synthesis) and RNA phosphoramidites with standard protectinggroups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeare purchased from (Promega). All phosphoramidites are used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichis used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes is used. The activator is 5-ethyl thiotetrazole(0.75M, American International Chemicals); for the PO-oxidationiodine/water/pyridine is used and for the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence is performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite are purchased from Biosearch Technologies. Conjugationof ligands to 5′-end and or internal position is achieved by usingappropriately protected ligand-phosphoramidite building block. Anextended 15 min coupling of 0.1 M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid-support-bound oligonucleotide. Oxidation of theinternucleotide phosphite to the phosphate is carried out using standardiodine-water as reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate is introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent. The cholesterol phosphoramidite is synthesized in house andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mLglass bottle (VWR). The oligonucleotide is cleaved from the support withsimultaneous deprotection of base and phosphate groups with 80 mL of amixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C.The bottle is cooled briefly on ice and then the ethanolic ammoniamixture is filtered into a new 250-mL bottle. The CPG is washed with2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixtureis then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dryice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionis then quenched with 50 mL of 20 mM sodium acetate and the pH isadjusted to 6.5. Oligonucleotide is stored in a freezer untilpurification.

Analysis

The oligonucleotides are analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phasepreparative HPLC. The unconjugated oligonucleotides are purified byanion-exchange HPLC on a TSK gel column packed in house. The buffers are20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodiumphosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides are pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides arediluted in water to 150 μL and then pipetted into special vials for CGEand LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

iRNA Preparation

For the general preparation of iRNA, equimolar amounts of sense andantisense strand are heated in 1×PBS at 95° C. for 5 min and slowlycooled to room temperature. Integrity of the duplex is confirmed by HPLCanalysis.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 2.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A adenosine Ccytidine G guanosine T thymidine U uridine N any nucleotide (G, A, C, Tor U) a 2′-O-methyladenosine c 2′-O-methylcytidine g2′-O-methylguanosine u 2′-O-methyruridine dT 2′-deoxythymidine sphosphorothioate linkage

Example 2 Mylip/Idol siRNA Design

Transcripts

siRNAs targeting Mylip/Idol were designed and synthesized. The designused human transcript NM_(—)013262.3 (SEQ ID NO: 644, FIG. 1) and rattranscript NM_(—)153789.3 (SEQ ID NO: 642) from the NCBI Refseqcollection.

siRNA duplexes were designed with 100% identity to the Mylip/Idol gene.

A total of 151 sense and 151 antisense human Mylip/Idol derived siRNAoligos were synthesized and formed into duplexes. The oligos arepresented in Tables 3 and 5 (human Mylip/Idol). In addition, 33 senseand 33 antisense rat Mylip/Idol derived siRNA oligos were synthesizedand formed into duplexes (see e.g., Tables 4 and 6).

TABLE 3 Sense and antisense strand sequences of human Mylip/Idol dsRNAsposition of 5′ base on Strand ID transcript Sequence with 3′ (S = sense;(NM_013262.3, SEQ dinucleotide SEQ AS = SEQ ID Sequence ID overhang IDantisense) NO: 644) (5′ to 3′) NO: (5′ to 3′) NO: S  240GCUGUGUUAUGUGACGAGG  20 GCUGUGUUAUGUGACGAGGNN   94 AS  240CCUCGUCACAUAACACAGC  21 CCUCGUCACAUAACACAGCNN   95 S  241CUGUGUUAUGUGACGAGGC  22 CUGUGUUAUGUGACGAGGCNN   96 AS  241GCCUCGUCACAUAACACAG  23 GCCUCGUCACAUAACACAGNN   97 S  244UGUUAUGUGACGAGGCCGG  24 UGUUAUGUGACGAGGCCGGNN   98 AS  244CCGGCCUCGUCACAUAACA  25 CCGGCCUCGUCACAUAACANN   99 S  245GUUAUGUGACGAGGCCGGA  26 GUUAUGUGACGAGGCCGGANN  100 AS  245UCCGGCCUCGUCACAUAAC  27 UCCGGCCUCGUCACAUAACNN  101 S  246UUAUGUGACGAGGCCGGAC  28 UUAUGUGACGAGGCCGGACNN  102 AS  246GUCCGGCCUCGUCACAUAA  29 GUCCGGCCUCGUCACAUAANN  103 S  247UAUGUGACGAGGCCGGACG  30 UAUGUGACGAGGCCGGACGNN  104 AS  247CGUCCGGCCUCGUCACAUA  31 CGUCCGGCCUCGUCACAUANN  105 S  248AUGUGACGAGGCCGGACGC  32 AUGUGACGAGGCCGGACGCNN  106 AS  248GCGUCCGGCCUCGUCACAU  33 GCGUCCGGCCUCGUCACAUNN  107 S  249UGUGACGAGGCCGGACGCG  34 UGUGACGAGGCCGGACGCGNN  108 AS  249CGCGUCCGGCCUCGUCACA  35 CGCGUCCGGCCUCGUCACANN  109 S  290AGGCGAAAGCCAACGGCGA  36 AGGCGAAAGCCAACGGCGANN  110 AS  290UCGCCGUUGGCUUUCGCCU  37 UCGCCGUUGGCUUUCGCCUNN  111 S  291GGCGAAAGCCAACGGCGAG  38 GGCGAAAGCCAACGGCGAGNN  112 AS  291CUCGCCGUUGGCUUUCGCC  39 CUCGCCGUUGGCUUUCGCCNN  113 S  331AGGCGACUGGGAAUCAUAG  40 AGGCGACUGGGAAUCAUAGNN  114 AS  331CUAUGAUUCCCAGUCGCCU  41 CUAUGAUUCCCAGUCGCCUNN  115 S  332GGCGACUGGGAAUCAUAGA  42 GGCGACUGGGAAUCAUAGANN  116 AS  332UCUAUGAUUCCCAGUCGCC  43 UCUAUGAUUCCCAGUCGCCNN  117 S  333GCGACUGGGAAUCAUAGAA  44 GCGACUGGGAAUCAUAGAANN  118 AS  333UUCUAUGAUUCCCAGUCGC  45 UUCUAUGAUUCCCAGUCGCNN  119 S  368UGCAGUUUACGGGUAGCAA  46 UGCAGUUUACGGGUAGCAANN  120 AS  368UUGCUACCCGUAAACUGCA  47 UUGCUACCCGUAAACUGCANN  121 S  369GCAGUUUACGGGUAGCAAA  48 GCAGUUUACGGGUAGCAAANN  122 AS  369UUUGCUACCCGUAAACUGC  49 UUUGCUACCCGUAAACUGCNN  123 S  370CAGUUUACGGGUAGCAAAG  50 CAGUUUACGGGUAGCAAAGNN  124 AS  370CUUUGCUACCCGUAAACUG  51 CUUUGCUACCCGUAAACUGNN  125 S  371AGUUUACGGGUAGCAAAGG  52 AGUUUACGGGUAGCAAAGGNN  126 AS  371CCUUUGCUACCCGUAAACU  53 CCUUUGCUACCCGUAAACUNN  127 S  372GUUUACGGGUAGCAAAGGU  54 GUUUACGGGUAGCAAAGGUNN  128 AS  372ACCUUUGCUACCCGUAAAC  55 ACCUUUGCUACCCGUAAACNN  129 S  373UUUACGGGUAGCAAAGGUG  56 UUUACGGGUAGCAAAGGUGNN  130 AS  373CACCUUUGCUACCCGUAAA  57 CACCUUUGCUACCCGUAAANN  131 S  386AAGGUGAAAGUUUAUGGCU  58 AAGGUGAAAGUUUAUGGCUNN  132 AS  386AGCCAUAAACUUUCACCUU  59 AGCCAUAAACUUUCACCUUNN  133 S  387AGGUGAAAGUUUAUGGCUA  60 AGGUGAAAGUUUAUGGCUANN  134 AS  387UAGCCAUAAACUUUCACCU  61 UAGCCAUAAACUUUCACCUNN  135 S  388GGUGAAAGUUUAUGGCUAA  62 GGUGAAAGUUUAUGGCUAANN  136 AS  388UUAGCCAUAAACUUUCACC  63 UUAGCCAUAAACUUUCACCNN  137 S  393AAGUUUAUGGCUAAACCUG  64 AAGUUUAUGGCUAAACCUGNN  138 AS  393CAGGUUUAGCCAUAAACUU  65 CAGGUUUAGCCAUAAACUUNN  139 S  395GUUUAUGGCUAAACCUGAG  66 GUUUAUGGCUAAACCUGAGNN  140 AS  395CUCAGGUUUAGCCAUAAAC  67 CUCAGGUUUAGCCAUAAACNN  141 S  434UGGAUGGGCUAGCCCCUUA  68 UGGAUGGGCUAGCCCCUUANN  142 AS  434UAAGGGGCUAGCCCAUCCA  69 UAAGGGGCUAGCCCAUCCANN  143 S  435GGAUGGGCUAGCCCCUUAC  70 GGAUGGGCUAGCCCCUUACNN  144 AS  435GUAAGGGGCUAGCCCAUCC  71 GUAAGGGGCUAGCCCAUCCNN  145 S  438UGGGCUAGCCCCUUACAGG  72 UGGGCUAGCCCCUUACAGGNN  146 AS  438CCUGUAAGGGGCUAGCCCA  73 CCUGUAAGGGGCUAGCCCANN  147 S  439GGGCUAGCCCCUUACAGGC  74 GGGCUAGCCCCUUACAGGCNN  148 AS  439GCCUGUAAGGGGCUAGCCC  75 GCCUGUAAGGGGCUAGCCCNN  149 S  440GGCUAGCCCCUUACAGGCU  76 GGCUAGCCCCUUACAGGCUNN  150 AS  440AGCCUGUAAGGGGCUAGCC  77 AGCCUGUAAGGGGCUAGCCNN  151 S  444AGCCCCUUACAGGCUUAAA  78 AGCCCCUUACAGGCUUAAANN  152 AS  444UUUAAGCCUGUAAGGGGCU  79 UUUAAGCCUGUAAGGGGCUNN  153 S  446CCCCUUACAGGCUUAAACU  80 CCCCUUACAGGCUUAAACUNN  154 AS  446AGUUUAAGCCUGUAAGGGG  81 AGUUUAAGCCUGUAAGGGGNN  155 S  498CUUACAGGAGCAGACUAGG  82 CUUACAGGAGCAGACUAGGNN  156 AS  498CCUAGUCUGCUCCUGUAAG  83 CCUAGUCUGCUCCUGUAAGNN  157 S  508CAGACUAGGCAUAUCUUUU  84 CAGACUAGGCAUAUCUUUUNN  158 AS  508AAAAGAUAUGCCUAGUCUG  85 AAAAGAUAUGCCUAGUCUGNN  159 S  640ACUGCCAAGUAUAACUAUG  86 ACUGCCAAGUAUAACUAUGNN  160 AS  640CAUAGUUAUACUUGGCAGU  87 CAUAGUUAUACUUGGCAGUNN  161 S  763UUGCAGAUUGUGUCGGCAA  88 UUGCAGAUUGUGUCGGCAANN  162 AS  763UUGCCGACACAAUCUGCAA  89 UUGCCGACACAAUCUGCAANN  163 S  764UGCAGAUUGUGUCGGCAAU  90 UGCAGAUUGUGUCGGCAAUNN  164 AS  764AUUGCCGACACAAUCUGCA  91 AUUGCCGACACAAUCUGCANN  165 S  765GCAGAUUGUGUCGGCAAUG  92 GCAGAUUGUGUCGGCAAUGNN  166 AS  765CAUUGCCGACACAAUCUGC  93 CAUUGCCGACACAAUCUGCNN  167 S  233CAGCCAUGCUGUGUUAUGU 648 CAGCCAUGCUGUGUUAUGUNN  876 AS  233ACAUAACACAGCAUGGCUG 649 ACAUAACACAGCAUGGCUGNN  877 S  330CAGGCGACUGGGAAUCAUA 650 CAGGCGACUGGGAAUCAUANN  878 AS  330UAUGAUUCCCAGUCGCCUG 651 UAUGAUUCCCAGUCGCCUGNN  879 S  335GACUGGGAAUCAUAGAAGU 652 GACUGGGAAUCAUAGAAGUNN  880 AS  335ACUUCUAUGAUUCCCAGUC 653 ACUUCUAUGAUUCCCAGUCNN  881 S  336ACUGGGAAUCAUAGAAGUU 654 ACUGGGAAUCAUAGAAGUUNN  882 AS  336AACUUCUAUGAUUCCCAGU 655 AACUUCUAUGAUUCCCAGUNN  883 S  341GAAUCAUAGAAGUUGACUA 656 GAAUCAUAGAAGUUGACUANN  884 AS  341UAGUCAACUUCUAUGAUUC 657 UAGUCAACUUCUAUGAUUCNN  885 S  404UAAACCUGAGAAACCGGAU 658 UAAACCUGAGAAACCGGAUNN  886 AS  404AUCCGGUUUCUCAGGUUUA 659 AUCCGGUUUCUCAGGUUUANN  887 S  454AGGCUUAAACUUAGAGUCA 660 AGGCUUAAACUUAGAGUCANN  888 AS  454UGACUCUAAGUUUAAGCCU 661 UGACUCUAAGUUUAAGCCUNN  889 S  455GGCUUAAACUUAGAGUCAA 662 GGCUUAAACUUAGAGUCAANN  890 AS  455UUGACUCUAAGUUUAAGCC 663 UUGACUCUAAGUUUAAGCCNN  891 S  501ACAGGAGCAGACUAGGCAU 664 ACAGGAGCAGACUAGGCAUNN  892 AS  501AUGCCUAGUCUGCUCCUGU 665 AUGCCUAGUCUGCUCCUGUNN  893 S  502CAGGAGCAGACUAGGCAUA 666 CAGGAGCAGACUAGGCAUANN  894 AS  502UAUGCCUAGUCUGCUCCUG 667 UAUGCCUAGUCUGCUCCUGNN  895 S  505GAGCAGACUAGGCAUAUCU 668 GAGCAGACUAGGCAUAUCUNN  896 AS  505AGAUAUGCCUAGUCUGCUC 669 AGAUAUGCCUAGUCUGCUCNN  897 S  507GCAGACUAGGCAUAUCUUU 670 GCAGACUAGGCAUAUCUUUNN  898 AS  507AAAGAUAUGCCUAGUCUGC 671 AAAGAUAUGCCUAGUCUGCNN  899 S  550UUGGCAGGCCACCUCUUGU 672 UUGGCAGGCCACCUCUUGUNN  900 AS  550ACAAGAGGUGGCCUGCCAA 673 ACAAGAGGUGGCCUGCCAANN  901 S  694UUGAACAGCAUUGUUGCAA 674 UUGAACAGCAUUGUUGCAANN  902 AS  694UUGCAACAAUGCUGUUCAA 675 UUGCAACAAUGCUGUUCAANN  903 S  746CAGCUGAAUACCAAGUUUU 676 CAGCUGAAUACCAAGUUUUNN  904 AS  746AAAACUUGGUAUUCAGCUG 677 AAAACUUGGUAUUCAGCUGNN  905 S  774GUCGGCAAUGGAAAACUAU 678 GUCGGCAAUGGAAAACUAUNN  906 AS  774AUAGUUUUCCAUUGCCGAC 679 AUAGUUUUCCAUUGCCGACNN  907 S  788ACUAUGGCAUAGAAUGGCA 680 ACUAUGGCAUAGAAUGGCANN  908 AS  788UGCCAUUCUAUGCCAUAGU 681 UGCCAUUCUAUGCCAUAGUNN  909 S  807UUCUGUGCGGGAUAGCGAA 682 UUCUGUGCGGGAUAGCGAANN  910 AS  807UUCGCUAUCCCGCACAGAA 683 UUCGCUAUCCCGCACAGAANN  911 S  850GGACCUGAAGGAAUCUCAA 684 GGACCUGAAGGAAUCUCAANN  912 AS  850UUGAGAUUCCUUCAGGUCC 685 UUGAGAUUCCUUCAGGUCCNN  913 S  873UAAAGAUGACUUUAGCCCA 686 UAAAGAUGACUUUAGCCCANN  914 AS  873UGGGCUAAAGUCAUCUUUA 687 UGGGCUAAAGUCAUCUUUANN  915 S  874AAAGAUGACUUUAGCCCAA 688 AAAGAUGACUUUAGCCCAANN  916 AS  874UUGGGCUAAAGUCAUCUUU 689 UUGGGCUAAAGUCAUCUUUNN  917 S  885UAGCCCAAUUAAUAGGAUA 690 UAGCCCAAUUAAUAGGAUANN  918 AS  885UAUCCUAUUAAUUGGGCUA 691 UAUCCUAUUAAUUGGGCUANN  919 S  889CCAAUUAAUAGGAUAGCUU 692 CCAAUUAAUAGGAUAGCUUNN  920 AS  889AAGCUAUCCUAUUAAUUGG 693 AAGCUAUCCUAUUAAUUGGNN  921 S  894UAAUAGGAUAGCUUAUCCU 694 UAAUAGGAUAGCUUAUCCUNN  922 AS  894AGGAUAAGCUAUCCUAUUA 695 AGGAUAAGCUAUCCUAUUANN  923 S  978CAGCAUCGUGCUCUUGUUU 696 CAGCAUCGUGCUCUUGUUUNN  924 AS  978AAACAAGAGCACGAUGCUG 697 AAACAAGAGCACGAUGCUGNN  925 S  981CAUCGUGCUCUUGUUUAAA 698 CAUCGUGCUCUUGUUUAAANN  926 AS  981UUUAAACAAGAGCACGAUG 699 UUUAAACAAGAGCACGAUGNN  927 S 1024GGGCUCUACCGAGCGAUAA 700 GGGCUCUACCGAGCGAUAANN  928 AS 1024UUAUCGCUCGGUAGAGCCC 701 UUAUCGCUCGGUAGAGCCCNN  929 S 1026GCUCUACCGAGCGAUAACA 702 GCUCUACCGAGCGAUAACANN  930 AS 1026UGUUAUCGCUCGGUAGAGC 703 UGUUAUCGCUCGGUAGAGCNN  931 S 1028UCUACCGAGCGAUAACAGA 704 UCUACCGAGCGAUAACAGANN  932 AS 1028UCUGUUAUCGCUCGGUAGA 705 UCUGUUAUCGCUCGGUAGANN  933 S 1030UACCGAGCGAUAACAGAGA 706 UACCGAGCGAUAACAGAGANN  934 AS 1030UCUCUGUUAUCGCUCGGUA 707 UCUCUGUUAUCGCUCGGUANN  935 S 1042ACAGAGACGCACGCAUUCU 708 ACAGAGACGCACGCAUUCUNN  936 AS 1042AGAAUGCGUGCGUCUCUGU 709 AGAAUGCGUGCGUCUCUGUNN  937 S 1113GAAGGGCCACUUGGCAUCU 710 GAAGGGCCACUUGGCAUCUNN  938 AS 1113AGAUGCCAAGUGGCCCUUC 711 AGAUGCCAAGUGGCCCUUCNN  939 S 1190CAUCAAAGGAGGUGUAUGA 712 CAUCAAAGGAGGUGUAUGANN  940 AS 1190UCAUACACCUCCUUUGAUG 713 UCAUACACCUCCUUUGAUGNN  941 S 1237GGCGUUGUGGACCUCGUUU 714 GGCGUUGUGGACCUCGUUUNN  942 AS 1237AAACGAGGUCCACAACGCC 715 AAACGAGGUCCACAACGCCNN  943 S 1240GUUGUGGACCUCGUUUCAA 716 GUUGUGGACCUCGUUUCAANN  944 AS 1240UUGAAACGAGGUCCACAAC 717 UUGAAACGAGGUCCACAACNN  945 S 1242UGUGGACCUCGUUUCAAGA 718 UGUGGACCUCGUUUCAAGANN  946 AS 1242UCUUGAAACGAGGUCCACA 719 UCUUGAAACGAGGUCCACANN  947 S 1279CACUCGCCUCUGAAGUCCU 720 CACUCGCCUCUGAAGUCCUNN  948 AS 1279AGGACUUCAGAGGCGAGUG 721 AGGACUUCAGAGGCGAGUGNN  949 S 1515GCAUGUCCAGCACGUCUAU 722 GCAUGUCCAGCACGUCUAUNN  950 AS 1515AUAGACGUGCUGGACAUGC 723 AUAGACGUGCUGGACAUGCNN  951 S 1517AUGUCCAGCACGUCUAUCU 724 AUGUCCAGCACGUCUAUCUNN  952 AS 1517AGAUAGACGUGCUGGACAU 725 AGAUAGACGUGCUGGACAUNN  953 S 1555CUCAAUCUGACUGUAAUCU 726 CUCAAUCUGACUGUAAUCUNN  954 AS 1555AGAUUACAGUCAGAUUGAG 727 AGAUUACAGUCAGAUUGAGNN  955 S 1557CAAUCUGACUGUAAUCUAA 728 CAAUCUGACUGUAAUCUAANN  956 AS 1557UUAGAUUACAGUCAGAUUG 729 UUAGAUUACAGUCAGAUUGNN  957 S 1558AAUCUGACUGUAAUCUAAU 730 AAUCUGACUGUAAUCUAAUNN  958 AS 1558AUUAGAUUACAGUCAGAUU 731 AUUAGAUUACAGUCAGAUUNN  959 S 1616UGCACUAUUAUAAACUAUU 732 UGCACUAUUAUAAACUAUUNN  960 AS 1616AAUAGUUUAUAAUAGUGCA 733 AAUAGUUUAUAAUAGUGCANN  961 S 1715AUAACACAGCUACUCCUCA 734 AUAACACAGCUACUCCUCANN  962 AS 1715UGAGGAGUAGCUGUGUUAU 735 UGAGGAGUAGCUGUGUUAUNN  963 S 1740AAACAUAUCCAUGCGUAGA 736 AAACAUAUCCAUGCGUAGANN  964 AS 1740UCUACGCAUGGAUAUGUUU 737 UCUACGCAUGGAUAUGUUUNN  965 S 1741AACAUAUCCAUGCGUAGAA 738 AACAUAUCCAUGCGUAGAANN  966 AS 1741UUCUACGCAUGGAUAUGUU 739 UUCUACGCAUGGAUAUGUUNN  967 S 1744AUAUCCAUGCGUAGAAUCA 740 AUAUCCAUGCGUAGAAUCANN  968 AS 1744UGAUUCUACGCAUGGAUAU 741 UGAUUCUACGCAUGGAUAUNN  969 S 1745UAUCCAUGCGUAGAAUCAA 742 UAUCCAUGCGUAGAAUCAANN  970 AS 1745UUGAUUCUACGCAUGGAUA 743 UUGAUUCUACGCAUGGAUANN  971 S 1753CGUAGAAUCAACAACUCCA 744 CGUAGAAUCAACAACUCCANN  972 AS 1753UGGAGUUGUUGAUUCUACG 745 UGGAGUUGUUGAUUCUACGNN  973 S 1837CUAGUAAAGGAAUAGGUAA 746 CUAGUAAAGGAAUAGGUAANN  974 AS 1837UUACCUAUUCCUUUACUAG 747 UUACCUAUUCCUUUACUAGNN  975 S 1838UAGUAAAGGAAUAGGUAAA 748 UAGUAAAGGAAUAGGUAAANN  976 AS 1838UUUACCUAUUCCUUUACUA 749 UUUACCUAUUCCUUUACUANN  977 S 1842AAAGGAAUAGGUAAAGUCU 750 AAAGGAAUAGGUAAAGUCUNN  978 AS 1842AGACUUUACCUAUUCCUUU 751 AGACUUUACCUAUUCCUUUNN  979 S 1843AAGGAAUAGGUAAAGUCUU 752 AAGGAAUAGGUAAAGUCUUNN  980 AS 1843AAGACUUUACCUAUUCCUU 753 AAGACUUUACCUAUUCCUUNN  981 S 1871UGAAGUGGCAACAUAGCCA 754 UGAAGUGGCAACAUAGCCANN  982 AS 1871UGGCUAUGUUGCCACUUCA 755 UGGCUAUGUUGCCACUUCANN  983 S 1872GAAGUGGCAACAUAGCCAA 756 GAAGUGGCAACAUAGCCAANN  984 AS 1872UUGGCUAUGUUGCCACUUC 757 UUGGCUAUGUUGCCACUUCNN  985 S 1893AGUUGGGUACCUUUUAGGA 758 AGUUGGGUACCUUUUAGGANN  986 AS 1893UCCUAAAAGGUACCCAACU 759 UCCUAAAAGGUACCCAACUNN  987 S 1915GAUGUUGUAAGUCUCCUUA 760 GAUGUUGUAAGUCUCCUUANN  988 AS 1915UAAGGAGACUUACAACAUC 761 UAAGGAGACUUACAACAUCNN  989 S 1921GUAAGUCUCCUUAAUGUAU 762 GUAAGUCUCCUUAAUGUAUNN  990 AS 1921AUACAUUAAGGAGACUUAC 763 AUACAUUAAGGAGACUUACNN  991 S 1933AAUGUAUCCUGAGGUAAGU 764 AAUGUAUCCUGAGGUAAGUNN  992 AS 1933ACUUACCUCAGGAUACAUU 765 ACUUACCUCAGGAUACAUUNN  993 S 1939UCCUGAGGUAAGUUUCCUA 766 UCCUGAGGUAAGUUUCCUANN  994 AS 1939UAGGAAACUUACCUCAGGA 767 UAGGAAACUUACCUCAGGANN  995 S 1954CCUACUGGCAGCAGAUUUU 768 CCUACUGGCAGCAGAUUUUNN  996 AS 1954AAAAUCUGCUGCCAGUAGG 769 AAAAUCUGCUGCCAGUAGGNN  997 S 2046UUUUGUAAAUUGUUGUCGU 770 UUUUGUAAAUUGUUGUCGUNN  998 AS 2046ACGACAACAAUUUACAAAA 771 ACGACAACAAUUUACAAAANN  999 S 2049UGUAAAUUGUUGUCGUUUU 772 UGUAAAUUGUUGUCGUUUUNN 1000 AS 2049AAAACGACAACAAUUUACA 773 AAAACGACAACAAUUUACANN 1001 S 2103GAUUGGAAGGCAAACAGGU 774 GAUUGGAAGGCAAACAGGUNN 1002 AS 2103ACCUGUUUGCCUUCCAAUC 775 ACCUGUUUGCCUUCCAAUCNN 1003 S 2109AAGGCAAACAGGUUUACAA 776 AAGGCAAACAGGUUUACAANN 1004 AS 2109UUGUAAACCUGUUUGCCUU 777 UUGUAAACCUGUUUGCCUUNN 1005 S 2159UGUUGUCAGAUUUAAACCA 778 UGUUGUCAGAUUUAAACCANN 1006 AS 2159UGGUUUAAAUCUGACAACA 779 UGGUUUAAAUCUGACAACANN 1007 S 2172AAACCAGUGUGGCUAGUAA 780 AAACCAGUGUGGCUAGUAANN 1008 AS 2172UUACUAGCCACACUGGUUU 781 UUACUAGCCACACUGGUUUNN 1009 S 2206AUGUGGGUGGCUCCCUAUU 782 AUGUGGGUGGCUCCCUAUUNN 1010 AS 2206AAUAGGGAGCCACCCACAU 783 AAUAGGGAGCCACCCACAUNN 1011 S 2248CCCCACAAGCCUUUCGAUU 784 CCCCACAAGCCUUUCGAUUNN 1012 AS 2248AAUCGAAAGGCUUGUGGGG 785 AAUCGAAAGGCUUGUGGGGNN 1013 S 2256GCCUUUCGAUUAUAAAAUA 786 GCCUUUCGAUUAUAAAAUANN 1014 AS 2256UAUUUUAUAAUCGAAAGGC 787 UAUUUUAUAAUCGAAAGGCNN 1015 S 2262CGAUUAUAAAAUACUACCA 788 CGAUUAUAAAAUACUACCANN 1016 AS 2262UGGUAGUAUUUUAUAAUCG 789 UGGUAGUAUUUUAUAAUCGNN 1017 S 2283CUUGUUAUAAGAUUACUGU 790 CUUGUUAUAAGAUUACUGUNN 1018 AS 2283ACAGUAAUCUUAUAACAAG 791 ACAGUAAUCUUAUAACAAGNN 1019 S 2293GAUUACUGUGGAGUAGUCA 792 GAUUACUGUGGAGUAGUCANN 1020 AS 2293UGACUACUCCACAGUAAUC 793 UGACUACUCCACAGUAAUCNN 1021 S 2296UACUGUGGAGUAGUCAAGU 794 UACUGUGGAGUAGUCAAGUNN 1022 AS 2296ACUUGACUACUCCACAGUA 795 ACUUGACUACUCCACAGUANN 1023 S 2428GUACAACUGAGGGUAGUUA 796 GUACAACUGAGGGUAGUUANN 1024 AS 2428UAACUACCCUCAGUUGUAC 797 UAACUACCCUCAGUUGUACNN 1025 S 2429UACAACUGAGGGUAGUUAA 798 UACAACUGAGGGUAGUUAANN 1026 AS 2429UUAACUACCCUCAGUUGUA 799 UUAACUACCCUCAGUUGUANN 1027 S 2431CAACUGAGGGUAGUUAACU 800 CAACUGAGGGUAGUUAACUNN 1028 AS 2431AGUUAACUACCCUCAGUUG 801 AGUUAACUACCCUCAGUUGNN 1029 S 2433ACUGAGGGUAGUUAACUCA 802 ACUGAGGGUAGUUAACUCANN 1030 AS 2433UGAGUUAACUACCCUCAGU 803 UGAGUUAACUACCCUCAGUNN 1031 S 2434CUGAGGGUAGUUAACUCAU 804 CUGAGGGUAGUUAACUCAUNN 1032 AS 2434AUGAGUUAACUACCCUCAG 805 AUGAGUUAACUACCCUCAGNN 1033 S 2436GAGGGUAGUUAACUCAUCA 806 GAGGGUAGUUAACUCAUCANN 1034 AS 2436UGAUGAGUUAACUACCCUC 807 UGAUGAGUUAACUACCCUCNN 1035 S 2438GGGUAGUUAACUCAUCACU 808 GGGUAGUUAACUCAUCACUNN 1036 AS 2438AGUGAUGAGUUAACUACCC 809 AGUGAUGAGUUAACUACCCNN 1037 S 2439GGUAGUUAACUCAUCACUU 810 GGUAGUUAACUCAUCACUUNN 1038 AS 2439AAGUGAUGAGUUAACUACC 811 AAGUGAUGAGUUAACUACCNN 1039 S 2441UAGUUAACUCAUCACUUCU 812 UAGUUAACUCAUCACUUCUNN 1040 AS 2441AGAAGUGAUGAGUUAACUA 813 AGAAGUGAUGAGUUAACUANN 1041 S 2489UGGUGUUGCUUUGCUUGAA 814 UGGUGUUGCUUUGCUUGAANN 1042 AS 2489UUCAAGCAAAGCAACACCA 815 UUCAAGCAAAGCAACACCANN 1043 S 2523AUAGCCUUACCAUAAGUAU 816 AUAGCCUUACCAUAAGUAUNN 1044 AS 2523AUACUUAUGGUAAGGCUAU 817 AUACUUAUGGUAAGGCUAUNN 1045 S 2530UACCAUAAGUAUUUAGAUA 818 UACCAUAAGUAUUUAGAUANN 1046 AS 2530UAUCUAAAUACUUAUGGUA 819 UAUCUAAAUACUUAUGGUANN 1047 S 2597AAGUAAGUGCUUAAGUAUU 820 AAGUAAGUGCUUAAGUAUUNN 1048 AS 2597AAUACUUAAGCACUUACUU 821 AAUACUUAAGCACUUACUUNN 1049 S 2610AGUAUUAACUUUGGGUUGU 822 AGUAUUAACUUUGGGUUGUNN 1050 AS 2610ACAACCCAAAGUUAAUACU 823 ACAACCCAAAGUUAAUACUNN 1051 S 2636GUAUGUUUCGAAGGGGUUU 824 GUAUGUUUCGAAGGGGUUUNN 1052 AS 2636AAACCCCUUCGAAACAUAC 825 AAACCCCUUCGAAACAUACNN 1053 S 2717CUGGUCAGCUAGCAGGUUU 826 CUGGUCAGCUAGCAGGUUUNN 1054 AS 2717AAACCUGCUAGCUGACCAG 827 AAACCUGCUAGCUGACCAGNN 1055 S 2718UGGUCAGCUAGCAGGUUUU 828 UGGUCAGCUAGCAGGUUUUNN 1056 AS 2718AAAACCUGCUAGCUGACCA 829 AAAACCUGCUAGCUGACCANN 1057 S 2720GUCAGCUAGCAGGUUUUCU 830 GUCAGCUAGCAGGUUUUCUNN 1058 AS 2720AGAAAACCUGCUAGCUGAC 831 AGAAAACCUGCUAGCUGACNN 1059 S 2740GGAUGUCGGGAGACCUAGA 832 GGAUGUCGGGAGACCUAGANN 1060 AS 2740UCUAGGUCUCCCGACAUCC 833 UCUAGGUCUCCCGACAUCCNN 1061 S 2741GAUGUCGGGAGACCUAGAU 834 GAUGUCGGGAGACCUAGAUNN 1062 AS 2741AUCUAGGUCUCCCGACAUC 835 AUCUAGGUCUCCCGACAUCNN 1063 S 2743UGUCGGGAGACCUAGAUGA 836 UGUCGGGAGACCUAGAUGANN 1064 AS 2743UCAUCUAGGUCUCCCGACA 837 UCAUCUAGGUCUCCCGACANN 1065 S 2768CGGGUGCAAUACUAGCUAA 838 CGGGUGCAAUACUAGCUAANN 1066 AS 2768UUAGCUAGUAUUGCACCCG 839 UUAGCUAGUAUUGCACCCGNN 1067 S 2771GUGCAAUACUAGCUAAGGU 840 GUGCAAUACUAGCUAAGGUNN 1068 AS 2771ACCUUAGCUAGUAUUGCAC 841 ACCUUAGCUAGUAUUGCACNN 1069 S 2772UGCAAUACUAGCUAAGGUA 842 UGCAAUACUAGCUAAGGUANN 1070 AS 2772UACCUUAGCUAGUAUUGCA 843 UACCUUAGCUAGUAUUGCANN 1071 S 2773GCAAUACUAGCUAAGGUAA 844 GCAAUACUAGCUAAGGUAANN 1072 AS 2773UUACCUUAGCUAGUAUUGC 845 UUACCUUAGCUAGUAUUGCNN 1073 S 2777UACUAGCUAAGGUAAAGCU 846 UACUAGCUAAGGUAAAGCUNN 1074 AS 2777AGCUUUACCUUAGCUAGUA 847 AGCUUUACCUUAGCUAGUANN 1075 S 2778ACUAGCUAAGGUAAAGCUA 848 ACUAGCUAAGGUAAAGCUANN 1076 AS 2778UAGCUUUACCUUAGCUAGU 849 UAGCUUUACCUUAGCUAGUNN 1077 S 2780UAGCUAAGGUAAAGCUAGA 850 UAGCUAAGGUAAAGCUAGANN 1078 AS 2780UCUAGCUUUACCUUAGCUA 851 UCUAGCUUUACCUUAGCUANN 1079 S 2852AAUGUAGCAGUAAUGUGUU 852 AAUGUAGCAGUAAUGUGUUNN 1080 AS 2852AACACAUUACUGCUACAUU 853 AACACAUUACUGCUACAUUNN 1081 S 2929GGCACAUAUUAGCAUAUAA 854 GGCACAUAUUAGCAUAUAANN 1082 AS 2929UUAUAUGCUAAUAUGUGCC 855 UUAUAUGCUAAUAUGUGCCNN 1083 S 2988AAAUAAUGUUUCCACGUAA 856 AAAUAAUGUUUCCACGUAANN 1084 AS 2988UUACGUGGAAACAUUAUUU 857 UUACGUGGAAACAUUAUUUNN 1085 S 2991UAAUGUUUCCACGUAAAGA 858 UAAUGUUUCCACGUAAAGANN 1086 AS 2991UCUUUACGUGGAAACAUUA 859 UCUUUACGUGGAAACAUUANN 1087 S 2992AAUGUUUCCACGUAAAGAA 860 AAUGUUUCCACGUAAAGAANN 1088 AS 2992UUCUUUACGUGGAAACAUU 861 UUCUUUACGUGGAAACAUUNN 1089 S 3006AAGAACUCUGUUAUAUCCU 862 AAGAACUCUGUUAUAUCCUNN 1090 AS 3006AGGAUAUAACAGAGUUCUU 863 AGGAUAUAACAGAGUUCUUNN 1091 S 3007AGAACUCUGUUAUAUCCUA 864 AGAACUCUGUUAUAUCCUANN 1092 AS 3007UAGGAUAUAACAGAGUUCU 865 UAGGAUAUAACAGAGUUCUNN 1093 S 3034UGUCUUUUAUAUUCGGGAU 866 UGUCUUUUAUAUUCGGGAUNN 1094 AS 3034AUCCCGAAUAUAAAAGACA 867 AUCCCGAAUAUAAAAGACANN 1095 S 3035GUCUUUUAUAUUCGGGAUA 868 GUCUUUUAUAUUCGGGAUANN 1096 AS 3035UAUCCCGAAUAUAAAAGAC 869 UAUCCCGAAUAUAAAAGACNN 1097 S 3036UCUUUUAUAUUCGGGAUAA 870 UCUUUUAUAUUCGGGAUAANN 1098 AS 3036UUAUCCCGAAUAUAAAAGA 871 UUAUCCCGAAUAUAAAAGANN 1099 S 3037CUUUUAUAUUCGGGAUAAU 872 CUUUUAUAUUCGGGAUAAUNN 1100 AS 3037AUUAUCCCGAAUAUAAAAG 873 AUUAUCCCGAAUAUAAAAGNN 1101 S 3049GGAUAAUAAAGACUUUAAA 874 GGAUAAUAAAGACUUUAAANN 1102 AS 3049UUUAAAGUCUUUAUUAUCC 875 UUUAAAGUCUUUAUUAUCCNN 1103

TABLE 4Sense and antisense strand sequences of mouse and rat Mylip/Idol dsRNAsposition of 5′ Strand ID base on (S = sense; transcript SEQSequence with 3′ SEQ AS = (NM_153789.3, ID dinucleotide overhang IDantisense) SEQ ID NO: 645) Sequence (5′ to 3′) NO: (5′ to 3′) NO: S   14GAGCGGCGCGGCCGUGUAG 168 GAGCGGCGCGGCCGUGUAGNN 234 AS   14CUACACGGCCGCGCCGCUC 169 CUACACGGCCGCGCCGCUCNN 235 S   26CGUGUAGCUCCCGGGAACU 170 CGUGUAGCUCCCGGGAACUNN 236 AS   26AGUUCCCGGGAGCUACACG 171 AGUUCCCGGGAGCUACACGNN 237 S  218GCUGUGCUAUGUGACGAGG 172 GCUGUGCUAUGUGACGAGGNN 238 AS   218CCUCGUCACAUAGCACAGC 173 CCUCGUCACAUAGCACAGCNN 239 S  220UGUGCUAUGUGACGAGGCC 174 UGUGCUAUGUGACGAGGCCNN 240 AS  220GGCCUCGUCACAUAGCACA 175 GGCCUCGUCACAUAGCACANN 241 S  485GCAGACAAGGCAUAUCUUU 176 GCAGACAAGGCAUAUCUUUNN 242 AS  485AAAGAUAUGCCUUGUCUGC 177 AAAGAUAUGCCUUGUCUGCNN 243 S  764GAACUACGGCAUAGAGUGG 178 GAACUACGGCAUAGAGUGGNN 244 AS  764CCACUCUAUGCCGUAGUUC 179 CCACUCUAUGCCGUAGUUCNN 245 S  766ACUACGGCAUAGAGUGGCA 180 ACUACGGCAUAGAGUGGCANN 246 AS  766UGCCACUCUAUGCCGUAGU 181 UGCCACUCUAUGCCGUAGUNN 247 S  857GGACUUUAGCCCUAUUAAC 182 GGACUUUAGCCCUAUUAACNN 248 AS  857GUUAAUAGGGCUAAAGUCC 183 GUUAAUAGGGCUAAAGUCCNN 249 S  858GACUUUAGCCCUAUUAACA 184 GACUUUAGCCCUAUUAACANN 250 AS  858UGUUAAUAGGGCUAAAGUC 185 UGUUAAUAGGGCUAAAGUCNN 251 S  867CCUAUUAACAGGAUAGCUU 186 CCUAUUAACAGGAUAGCUUNN 252 AS  867AAGCUAUCCUGUUAAUAGG 187 AAGCUAUCCUGUUAAUAGGNN 253 S  869UAUUAACAGGAUAGCUUAU 188 UAUUAACAGGAUAGCUUAUNN 254 AS  869AUAAGCUAUCCUGUUAAUA 189 AUAAGCUAUCCUGUUAAUANN 255 S  870AUUAACAGGAUAGCUUAUC 190 AUUAACAGGAUAGCUUAUCNN 256 AS  870GAUAAGCUAUCCUGUUAAU 191 GAUAAGCUAUCCUGUUAAUNN 257 S  871UUAACAGGAUAGCUUAUCC 192 UUAACAGGAUAGCUUAUCCNN 258 AS  871GGAUAAGCUAUCCUGUUAA 193 GGAUAAGCUAUCCUGUUAANN 259 S  873AACAGGAUAGCUUAUCCUG 194 AACAGGAUAGCUUAUCCUGNN 260 AS  873CAGGAUAAGCUAUCCUGUU 195 CAGGAUAAGCUAUCCUGUUNN 261 S  875CAGGAUAGCUUAUCCUGUG 196 CAGGAUAGCUUAUCCUGUGNN 262 AS  875CACAGGAUAAGCUAUCCUG 197 CACAGGAUAAGCUAUCCUGNN 263 S  919AGAAUGUCUACUUGACCGU 198 AGAAUGUCUACUUGACCGUNN 264 AS  919ACGGUCAAGUAGACAUUCU 199 ACGGUCAAGUAGACAUUCUNN 265 S   92UGUCUACUUGACCGUCACC 200 UGUCUACUUGACCGUCACCNN 266 AS  923GGUGACGGUCAAGUAGACA 201 GGUGACGGUCAAGUAGACANN 267 S 1815UCAAGUAAAGGAGUAGAUA 202 UCAAGUAAAGGAGUAGAUANN 268 AS 1815UAUCUACUCCUUUACUUGA 203 UAUCUACUCCUUUACUUGANN 269 S 1856GGCAACAUGGCCCAACCGU 204 GGCAACAUGGCCCAACCGUNN 270 AS 1856ACGGUUGGGCCAUGUUGCC 205 ACGGUUGGGCCAUGUUGCCNN 271 S 1859AACAUGGCCCAACCGUGGG 206 AACAUGGCCCAACCGUGGGNN 272 AS 1859CCCACGGUUGGGCCAUGUU 207 CCCACGGUUGGGCCAUGUUNN 273 S 1861CAUGGCCCAACCGUGGGCA 208 CAUGGCCCAACCGUGGGCANN 274 AS 1861UGCCCACGGUUGGGCCAUG 209 UGCCCACGGUUGGGCCAUGNN 275 S 1968UUGUAUGGUCAUGGAGCGC 210 UUGUAUGGUCAUGGAGCGCNN 276 AS 1968GCGCUCCAUGACCAUACAA 211 GCGCUCCAUGACCAUACAANN 277 S 1969UGUAUGGUCAUGGAGCGCU 212 UGUAUGGUCAUGGAGCGCUNN 278 AS 1969AGCGCUCCAUGACCAUACA 213 AGCGCUCCAUGACCAUACANN 279 S 2512UCUACAGCCUUAUAGGUUU 214 UCUACAGCCUUAUAGGUUUNN 280 AS 2512AAACCUAUAAGGCUGUAGA 215 AAACCUAUAAGGCUGUAGANN 281 S 2695GAAGCUAGUGAGCUAGGGG 216 GAAGCUAGUGAGCUAGGGGNN 282 AS 2695CCCCUAGCUCACUAGCUUC 217 CCCCUAGCUCACUAGCUUCNN 283 S 2744CCUCAUCGGGUGCAAUACU 218 CCUCAUCGGGUGCAAUACUNN 284 AS 2744AGUAUUGCACCCGAUGAGG 219 AGUAUUGCACCCGAUGAGGNN 285 S 2745CUCAUCGGGUGCAAUACUA 220 CUCAUCGGGUGCAAUACUANN 286 AS 2745UAGUAUUGCACCCGAUGAG 221 UAGUAUUGCACCCGAUGAGNN 287 S 2746UCAUCGGGUGCAAUACUAG 222 UCAUCGGGUGCAAUACUAGNN 288 AS 2746CUAGUAUUGCACCCGAUGA 223 CUAGUAUUGCACCCGAUGANN 289 S 2747CAUCGGGUGCAAUACUAGC 224 CAUCGGGUGCAAUACUAGCNN 290 AS 2747GCUAGUAUUGCACCCGAUG 225 GCUAGUAUUGCACCCGAUGNN 291 S 2748AUCGGGUGCAAUACUAGCU 226 AUCGGGUGCAAUACUAGCUNN 292 AS 2748AGCUAGUAUUGCACCCGAU 227 AGCUAGUAUUGCACCCGAUNN 293 S 2749UCGGGUGCAAUACUAGCUA 228 UCGGGUGCAAUACUAGCUANN 294 AS 2749UAGCUAGUAUUGCACCCGA 229 UAGCUAGUAUUGCACCCGANN 295 S 2918AUUAGCAUAUAAGCCUUUA 230 AUUAGCAUAUAAGCCUUUANN 296 AS 2918UAAAGGCUUAUAUGCUAAU 231 UAAAGGCUUAUAUGCUAAUNN 297 S 2919UUAGCAUAUAAGCCUUUAU 232 UUAGCAUAUAAGCCUUUAUNN 298 AS 2919AUAAAGGCUUAUAUGCUAA 233 AUAAAGGCUUAUAUGCUAANN 299

TABLE 5 Sense and antisense strand sequences of human Mylip/Idol dsRNAsposition of 5′ Sequence with Sequence with Strand ID base on3′deoxythimidine 3′deoxythimidine (S = sense; transcript overhang SEQoverhang SEQ AS = (NM_013262.3, (phosphodiester ID (phosphorothioate IDantisense) SEQ ID NO: 644) linkage)(5′ to 3′) NO: linkage)(5′ to 3′) NO:S 240 GCUGUGUUAUGUGACGAGGdT 300 GCUGUGUUAUGUGACGAGGdTs 374 dT dT AS 240CCUCGUCACAUAACACAGCdT 301 CCUCGUCACAUAACACAGCdTs 375 dT dT S 241CUGUGUUAUGUGACGAGGCdT 302 CUGUGUUAUGUGACGAGGdTs 376 dT dTC AS 241GCCUCGUCACAUAACACAGdT 303 GCCUCGUCACAUAACACAGdTs 377 dT dT S 244UGUUAUGUGACGAGGCCGGdT 304 UGUUAUGUGACGAGGCCGGdTs 378 dT dT AS 244CCGGCCUCGUCACAUAACAdT 305 CCGGCCUCGUCACAUAACAdTs 379 dT dT S 245GUUAUGUGACGAGGCCGGAdT 306 GUUAUGUGACGAGGCCGGAdTs 380 dT dT AS 245UCCGGCCUCGUCACAUAACdT 307 UCCGGCCUCGUCACAUAACdTs 381 dT dT S 246UUAUGUGACGAGGCCGGACdT 308 UUAUGUGACGAGGCCGGACdTs 382 dT dT AS 246GUCCGGCCUCGUCACAUAAdT 309 GUCCGGCCUCGUCACAUAAdTs 383 dT dT S 247UAUGUGACGAGGCCGGACGdT 310 UAUGUGACGAGGCCGGACGdTs 384 dT dT AS 247CGUCCGGCCUCGUCACAUAdT 311 CGUCCGGCCUCGUCACAUAdTs 385 dT dT S 248AUGUGACGAGGCCGGACGCdT 312 AUGUGACGAGGCCGGACGCdTs 386 dT dT AS 248GCGUCCGGCCUCGUCACAUdT 313 GCGUCCGGCCUCGUCACAUdTs 387 dT dT S 249UGUGACGAGGCCGGACGCGdT 314 UGUGACGAGGCCGGACGCGdTs 388 dT dT AS 249CGCGUCCGGCCUCGUCACAdT 315 CGCGUCCGGCCUCGUCACAdTs 389 dT dT S 290AGGCGAAAGCCAACGGCGAdT 316 AGGCGAAAGCCAACGGCGAdTs 390 dT dT AS 290UCGCCGUUGGCUUUCGCCUdT 317 UCGCCGUUGGCUUUCGCCUdTs 391 dT dT S 291GGCGAAAGCCAACGGCGAGdT 318 GGCGAAAGCCAACGGCGAGdTs 392 dT dT AS 291CUCGCCGUUGGCUUUCGCCdT 319 CUCGCCGUUGGCUUUCGCCdTs 393 dT dT S 331AGGCGACUGGGAAUCAUAGdT 320 AGGCGACUGGGAAUCAUAGdTs 394 dT dT AS 331CUAUGAUUCCCAGUCGCCUdT 321 CUAUGAUUCCCAGUCGCCUdTs 395 dT dT S 332GGCGACUGGGAAUCAUAGAdT 322 GGCGACUGGGAAUCAUAGAdTs 396 dT dT AS 332UCUAUGAUUCCCAGUCGCCdTs 323 UCUAUGAUUCCCAGUCGCC 397 dT S 333GCGACUGGGAAUCAUAGAAdT 324 GCGACUGGGAAUCAUAGAAdTs 398 dT dT AS 333UUCUAUGAUUCCCAGUCGCdT 325 UUCUAUGAUUCCCAGUCGCdTs 399 dT dT S 368UGCAGUUUACGGGUAGCAAdT 326 UGCAGUUUACGGGUAGCAAdTs 400 dT dT AS 368UUGCUACCCGUAAACUGCAdT 327 UUGCUACCCGUAAACUGCAdTs 401 dT dT S 369GCAGUUUACGGGUAGCAAAdT 328 GCAGUUUACGGGUAGCAAAdTs 402 dT dT AS 369UUUGCUACCCGUAAACUGCdT 329 UUUGCUACCCGUAAACUGCdTs 403 dT dT S 370CAGUUUACGGGUAGCAAAGdT 330 CAGUUUACGGGUAGCAAAGdTs 404 dT dT AS 370CUUUGCUACCCGUAAACUGdT 331 CUUUGCUACCCGUAAACUGdTs 405 dT dT S 371AGUUUACGGGUAGCAAAGGdT 332 AGUUUACGGGUAGCAAAGGdTs 406 dT dT AS 371CCUUUGCUACCCGUAAACUdT 333 CCUUUGCUACCCGUAAACUdTs 407 dT dT S 372GUUUACGGGUAGCAAAGGUdT 334 GUUUACGGGUAGCAAAGGUdTs 408 dT dT AS 372ACCUUUGCUACCCGUAAACdT 335 ACCUUUGCUACCCGUAAACdTs 409 dT dT S 373UUUACGGGUAGCAAAGGUGdT 336 UUUACGGGUAGCAAAGGUGdTs 410 dT dT AS 373CACCUUUGCUACCCGUAAAdT 337 CACCUUUGCUACCCGUAAAdTs 411 dT dT S 386AAGGUGAAAGUUUAUGGCUdT 338 AAGGUGAAAGUUUAUGGCUdTs 412 dT dT AS 386AGCCAUAAACUUUCACCUUdT 339 AGCCAUAAACUUUCACCUUdTs 413 dT dT S 387AGGUGAAAGUUUAUGGCUAdT 340 AGGUGAAAGUUUAUGGCUAdTs 414 dT dT AS 387UAGCCAUAAACUUUCACCUdT 341 UAGCCAUAAACUUUCACCUdTs 415 dT dT S 388GGUGAAAGUUUAUGGCUAAdT 342 GGUGAAAGUUUAUGGCUAAdTs 416 dT dT AS 388UUAGCCAUAAACUUUCACCdT 343 UUAGCCAUAAACUUUCACCdTs 417 dT dT S 393AAGUUUAUGGCUAAACCUGdT 344 AAGUUUAUGGCUAAACCUGdTs 418 dT dT AS 393CAGGUUUAGCCAUAAACUUdT 345 CAGGUUUAGCCAUAAACUUdTs 419 dT dT S 395GUUUAUGGCUAAACCUGAGdT 346 GUUUAUGGCUAAACCUGAGdTs 420 dT dT AS 395CUCAGGUUUAGCCAUAAACdT 347 CUCAGGUUUAGCCAUAAACdTs 421 dT dT S 434UGGAUGGGCUAGCCCCUUAdT 348 UGGAUGGGCUAGCCCCUUAdTs 422 dT dT AS 434UAAGGGGCUAGCCCAUCCAdT 349 UAAGGGGCUAGCCCAUCCAdTs 423 dT dT S 435GGAUGGGCUAGCCCCUUACdT 350 GGAUGGGCUAGCCCCUUACdTs 424 dT dT AS 435GUAAGGGGCUAGCCCAUCCdT 351 GUAAGGGGCUAGCCCAUCCdTs 425 dT dT S 438UGGGCUAGCCCCUUACAGGdT 352 UGGGCUAGCCCCUUACAGGdTs 426 dT dT AS 438CCUGUAAGGGGCUAGCCCAdT 353 CCUGUAAGGGGCUAGCCCAdTs 427 dT dT S 439GGGCUAGCCCCUUACAGGCdT 354 GGGCUAGCCCCUUACAGGCdTs 428 dT dT AS 439GCCUGUAAGGGGCUAGCCCdT 355 GCCUGUAAGGGGCUAGCCCdTs 429 dT dT S 440GGCUAGCCCCUUACAGGCUdT 356 GGCUAGCCCCUUACAGGCUdTs 430 dT dT AS 440AGCCUGUAAGGGGCUAGCCdT 357 AGCCUGUAAGGGGCUAGCCdTs 431 dT dT S 444AGCCCCUUACAGGCUUAAAdT 358 AGCCCCUUACAGGCUUAAAdTs 432 dT dT AS 444UUUAAGCCUGUAAGGGGCUdT 359 UUUAAGCCUGUAAGGGGCUdTs 433 dT dT S 446CCCCUUACAGGCUUAAACUdT 360 CCCCUUACAGGCUUAAACUdTs 434 dT dT AS 446AGUUUAAGCCUGUAAGGGGdT 361 AGUUUAAGCCUGUAAGGGGdTs 435 dT dT S 498CUUACAGGAGCAGACUAGGdT 362 CUUACAGGAGCAGACUAGGdTs 436 dT dT AS 498CCUAGUCUGCUCCUGUAAGdT 363 CCUAGUCUGCUCCUGUAAGdTs 437 dT dT S 508CAGACUAGGCAUAUCUUUUdT 364 CAGACUAGGCAUAUCUUUUdTs 438 dT dT AS 508AAAAGAUAUGCCUAGUCUGdT 365 AAAAGAUAUGCCUAGUCUGdTs 439 dT dT S 640ACUGCCAAGUAUAACUAUGdT 366 ACUGCCAAGUAUAACUAUGdTs 440 dT dT AS 640CAUAGUUAUACUUGGCAGUdT 367 CAUAGUUAUACUUGGCAGUdTs 441 dT dT S 763UUGCAGAUUGUGUCGGCAAdT 368 UUGCAGAUUGUGUCGGCAAdTs 442 dT dT AS 763UUGCCGACACAAUCUGCAAdT 369 UUGCCGACACAAUCUGCAAdTs 443 dT dT S 764UGCAGAUUGUGUCGGCAAUdT 370 UGCAGAUUGUGUCGGCAAUdTs 444 dT dT AS 764AUUGCCGACACAAUCUGCAdT 371 AUUGCCGACACAAUCUGCAdTs 445 dT dT S 765GCAGAUUGUGUCGGCAAUGdT 372 GCAGAUUGUGUCGGCAAUGdTs 446 dT dT AS 765CAUUGCCGACACAAUCUGCdT 373 CAUUGCCGACACAAUCUGCdTs 447 dT dT

TABLE 6Sense and antisense strand sequences of mouse/rat Mylip/Idol dsRNAsposition of 5′ Sequence with Sequence with Strand ID base on3′deoxythimidine 3′deoxythimidine (S = sense; transcript overhang SEQoverhang SEQ AS = (NM_153789.3, (phosphodiester ID (phosphorothioate IDantisense) SEQ ID NO: 645) linkage)(5′ to 3′) NO: linkage)(5′ to 3′) NO:S   14 GAGCGGCGCGGCCGUGUAGdT 448 GAGCGGCGCGGCCGUGUAGdTs 514 dT dT AS  14 CUACACGGCCGCGCCGCUCdT 449 CUACACGGCCGCGCCGCUCdTs 515 dT dT S   26CGUGUAGCUCCCGGGAACUdT 450 CGUGUAGCUCCCGGGAACUdTs 516 dT dT AS   26AGUUCCCGGGAGCUACACGdT 451 AGUUCCCGGGAGCUACACGdTs 517 dT dT S  218GCUGUGCUAUGUGACGAGGdT 452 GCUGUGCUAUGUGACGAGGdTs 518 dT dT AS  218CCUCGUCACAUAGCACAGCdT 453 CCUCGUCACAUAGCACAGCdTs 519 dT dT S  220UGUGCUAUGUGACGAGGCCdT 454 UGUGCUAUGUGACGAGGCCdTs 520 dT dT AS  220GGCCUCGUCACAUAGCACAdT 455 GGCCUCGUCACAUAGCACAdTs 521 dT dT S  485GCAGACAAGGCAUAUCUUUdT 456 GCAGACAAGGCAUAUCUUUdTs 522 dT dT AS  485AAAGAUAUGCCUUGUCUGCdT 457 AAAGAUAUGCCUUGUCUGCdTs 523 dT dT S  764GAACUACGGCAUAGAGUGGdT 458 GAACUACGGCAUAGAGUGGdTs 524 dT dT AS  764CCACUCUAUGCCGUAGUUCdT 459 CCACUCUAUGCCGUAGUUCdTs 525 dT dT S  766ACUACGGCAUAGAGUGGCAdT 460 ACUACGGCAUAGAGUGGCAdTs 526 dT dT AS  766UGCCACUCUAUGCCGUAGUdT 461 UGCCACUCUAUGCCGUAGUdTs 527 dT dT S  857GGACUUUAGCCCUAUUAACdT 462 GGACUUUAGCCCUAUUAACdTs 528 dT dT AS  857GUUAAUAGGGCUAAAGUCCdT 463 GUUAAUAGGGCUAAAGUCCdTs 529 dT dT S  858GACUUUAGCCCUAUUAACAdT 464 GACUUUAGCCCUAUUAACAdTs 530 dT dT AS  858UGUUAAUAGGGCUAAAGUCdT 465 UGUUAAUAGGGCUAAAGUCdTs 531 dT dT S  867CCUAUUAACAGGAUAGCUUdT 466 CCUAUUAACAGGAUAGCUUdTs 532 dT dT AS  867AAGCUAUCCUGUUAAUAGGdT 467 AAGCUAUCCUGUUAAUAGGdTs 533 dT dT S  869UAUUAACAGGAUAGCUUAUdT 468 UAUUAACAGGAUAGCUUAUdTs 534 dT dT AS  869AUAAGCUAUCCUGUUAAUAdT 469 AUAAGCUAUCCUGUUAAUAdTs 535 dT dT S  870AUUAACAGGAUAGCUUAUCdT 470 AUUAACAGGAUAGCUUAUCdTs 536 dT dT AS  870GAUAAGCUAUCCUGUUAAUdT 471 GAUAAGCUAUCCUGUUAAUdTs 537 dT dT S  871UUAACAGGAUAGCUUAUCCdT 472 UUAACAGGAUAGCUUAUCCdTs 538 dT dT AS  871GGAUAAGCUAUCCUGUUAAdT 473 GGAUAAGCUAUCCUGUUAAdTs 539 dT dT S  873AACAGGAUAGCUUAUCCUGdT 474 AACAGGAUAGCUUAUCCUGdTs 540 dT dT AS  873CAGGAUAAGCUAUCCUGUUdT 475 CAGGAUAAGCUAUCCUGUUdTs 541 dT dT S  875CAGGAUAGCUUAUCCUGUGdT 476 CAGGAUAGCUUAUCCUGUGdTs 542 dT dT AS  875CACAGGAUAAGCUAUCCUGdT 477 CACAGGAUAAGCUAUCCUGdTs 543 dT dT S  919AGAAUGUCUACUUGACCGUdT 478 AGAAUGUCUACUUGACCGUdTs 544 dT dT AS  919ACGGUCAAGUAGACAUUCUdT 479 ACGGUCAAGUAGACAUUCUdTs 545 dT dT S   92UGUCUACUUGACCGUCACCdT 480 UGUCUACUUGACCGUCACCdTs 546 dT dT AS  923GGUGACGGUCAAGUAGACAdT 481 GGUGACGGUCAAGUAGACAdTs 547 dT dT S 1815UCAAGUAAAGGAGUAGAUAdT 482 UCAAGUAAAGGAGUAGAUAdTs 548 dT dT AS 1815UAUCUACUCCUUUACUUGAdT 483 UAUCUACUCCUUUACUUGAdTs 549 dT dT S 1856GGCAACAUGGCCCAACCGUdT 484 GGCAACAUGGCCCAACCGUdTs 550 dT dT AS 1856ACGGUUGGGCCAUGUUGCCdT 485 ACGGUUGGGCCAUGUUGCCdTs 551 dT dT S 1859AACAUGGCCCAACCGUGGGdT 486 AACAUGGCCCAACCGUGGGdTs 552 dT dT AS 1859CCCACGGUUGGGCCAUGUUdT 487 CCCACGGUUGGGCCAUGUUdTs 553 dT dT S 1861CAUGGCCCAACCGUGGGCAdT 488 CAUGGCCCAACCGUGGGCAdTs 554 dT dT AS 1861UGCCCACGGUUGGGCCAUGdT 489 UGCCCACGGUUGGGCCAUGdTs 555 dT dT S 1968UUGUAUGGUCAUGGAGCGCdT 490 UUGUAUGGUCAUGGAGCGCdTs 556 dT dT AS 1968GCGCUCCAUGACCAUACAAdT 491 GCGCUCCAUGACCAUACAAdTs 557 dT dT S 1969UGUAUGGUCAUGGAGCGCUdT 492 UGUAUGGUCAUGGAGCGCUdTs 558 dT dT AS 1969AGCGCUCCAUGACCAUACAdT 493 AGCGCUCCAUGACCAUACAdTs 559 dT dT S 2512UCUACAGCCUUAUAGGUUUdT 494 UCUACAGCCUUAUAGGUUUdTs 560 dT dT AS 2512AAACCUAUAAGGCUGUAGAdT 495 AAACCUAUAAGGCUGUAGAdTs 561 dT dT S 2695GAAGCUAGUGAGCUAGGGGdT 496 GAAGCUAGUGAGCUAGGGGdTs 562 dT dT AS 2695CCCCUAGCUCACUAGCUUCdT 497 CCCCUAGCUCACUAGCUUCdTs 563 dT dT S 2744CCUCAUCGGGUGCAAUACUdT 498 CCUCAUCGGGUGCAAUACUdTs 564 dT dT AS 2744AGUAUUGCACCCGAUGAGGdT 499 AGUAUUGCACCCGAUGAGGdTs 565 dT dT S 2745CUCAUCGGGUGCAAUACUAdT 500 CUCAUCGGGUGCAAUACUAdTs 566 dT dT AS 2745UAGUAUUGCACCCGAUGAGdT 501 UAGUAUUGCACCCGAUGAGdTs 567 dT dT S 2746UCAUCGGGUGCAAUACUAGdT 502 UCAUCGGGUGCAAUACUAGdTs 568 dT dT AS 2746CUAGUAUUGCACCCGAUGAdT 503 CUAGUAUUGCACCCGAUGAdTs 569 dT dT S 2747CAUCGGGUGCAAUACUAGCdT 504 CAUCGGGUGCAAUACUAGCdTs 570 dT dT AS 2747GCUAGUAUUGCACCCGAUGdT 505 GCUAGUAUUGCACCCGAUGdTs 571 dT dT S 2748AUCGGGUGCAAUACUAGCUdT 506 AUCGGGUGCAAUACUAGCUdTs 572 dT dT AS 2748AGCUAGUAUUGCACCCGAUdT 507 AGCUAGUAUUGCACCCGAUdTs 573 dT dT S 2749UCGGGUGCAAUACUAGCUAdT 508 UCGGGUGCAAUACUAGCUAdTs 574 dT dT AS 2749UAGCUAGUAUUGCACCCGAdT 509 UAGCUAGUAUUGCACCCGAdTs 575 dT dT S 2918AUUAGCAUAUAAGCCUUUAdT 510 AUUAGCAUAUAAGCCUUUAdTs 576 dT dT AS 2918UAAAGGCUUAUAUGCUAAUdT 511 UAAAGGCUUAUAUGCUAAUdTs 577 dT dT S 2919UUAGCAUAUAAGCCUUUAUdT 512 UUAGCAUAUAAGCCUUUAUdTs 578 dT dT AS 2919AUAAAGGCUUAUAUGCUAAdT 513 AUAAAGGCUUAUAUGCUAAdTs 579 dT dT

TABLE 7 Chemically modified sense and antisensestrand sequences of Mylip/Idol dsRNAs Position of Strand 5′ base onID (S = transcript sense; (NM_ AS = 001098791.1, SEQ anti- SEQ ID IDsense) NO: 1299) Sequence (5′ to 3′) NO: S GAGcGGcGcGGccGuGuAGdTsdT 580AS CuAcACGGCCGCGCCGCUCdTsdT 581 S cGuGuAGcucccGGGAAcudTsdT 582 ASAGUUCCCGGGAGCuAcACGdTsdT 583 S GcuGuGcuAuGuGAcGAGGdTsdT 584 ASCCUCGUcAcAuAGcAcAGCdTsdT 585 S uGuGcuAuGuGAcGAGGccdTsdT 586 ASGGCCUCGUcAcAuAGcAcAdTsdT 587 S GcAGAcAAGGcAuAucuuudTsdT 588 ASAAAGAuAUGCCUUGUCUGCdTsdT 589 S GAAcuAcGGcAuAGAGuGGdTsdT 590 ASCcACUCuAUGCCGuAGUUCdTsdT 591 S AcuAcGGcAuAGAGuGGcAdTsdT 592 ASUGCcACUCuAUGCCGuAGUdTsdT 593 S GGAcuuuAGcccuAuuAAcdTsdT 594 ASGUuAAuAGGGCuAAAGUCCdTsdT 595 S GAcuuuAGcccuAuuAAcAdTsdT 596 ASUGUuAAuAGGGCuAAAGUCdTsdT 597 S ccuAuuAAcAGGAuAGcuudTsdT 598 ASAAGCuAUCCUGUuAAuAGGdTsdT 599 S uAuuAAcAGGAuAGcuuAudTsdT 600 ASAuAAGCuAUCCUGUuAAuAdTsdT 601 S AuuAAcAGGAuAGcuuAucdTsdT 602 ASGAuAAGCuAUCCUGUuAAUdTsdT 603 S uuAAcAGGAuAGcuuAuccdTsdT 604 ASGGAuAAGCuAUCCUGUuAAdTsdT 605 S cAGGAuAGcuuAuccuGuGdTsdT 606 AScAcAGGAuAAGCuAUCCUGdTsdT 607 S AGAAuGucuAcuuGAccGudTsdT 608 ASACGGUcAAGuAGAcAUUCUdTsdT 609 S uGucuAcuuGAccGucAccdTsdT 610 ASGGUGACGGUcAAGuAGAcAdTsdT 611 S ucAAGuAAAGGAGuAGAuAdTsdT 612 ASuAUCuACUCCUUuACUUGAdTsdT 613 S GGcAAcAuGGcccAAccGudTsdT 614 ASACGGUUGGGCcAUGUUGCCdTsdT 615 S AAcAuGGcccAAccGuGGGdTsdT 616 ASCCcACGGUUGGGCcAUGUUdTsdT 617 S cAuGGcccAAccGuGGGcAdTsdT 618 ASUGCCcACGGUUGGGCcAUGdTsdT 619 S uuGuAuGGucAuGGAGcGcdTsdT 620 ASGCGCUCcAUGACcAuAcAAdTsdT 621 S uGuAuGGucAuGGAGcGcudTsdT 622 ASAGCGCUCcAUGACcAuAcAdTsdT 623 S ucuAcAGccuuAuAGGuuudTsdT 624 ASAAACCuAuAAGGCUGuAGAdTsdT 625 S GAAGcuAGuGAGcuAGGGGdTsdT 626 ASCCCCuAGCUcACuAGCUUCdTsdT 627 S ccucAucGGGuGcAAuAcudTsdT 628 ASAGuAUUGcACCCGAUGAGGdTsdT 629 S cucAucGGGuGcAAuAcuAdTsdT 630 ASuAGuAUUGcACCCGAUGAGdTsdT 631 S ucAucGGGuGcAAuAcuAGdTsdT 632 ASCuAGuAUUGcACCCGAUGAdTsdT 633 S cAucGGGuGcAAuAcuAGcdTsdT 634 ASGCuAGuAUUGcACCCGAUGdTsdT 635 S AucGGGuGcAAuAcuAGcudTsdT 636 ASAGCuAGuAUUGcACCCGAUdTsdT 637 S ucGGGuGcAAuAcuAGcuAdTsdT 638 ASuAGCuAGuAUUGcACCCGAdTsdT 639 S AuuAGcAuAuAAGccuuuAdTsdT 640 ASuAAAGGCUuAuAUGCuAAUdTsdT 641 S AAcAGGAuAGcuuAuccuGdTsdT 642 AScAGGAuAAGCuAUCCUGUUdTsdT 643

Synthesis of Mylip/Idol Sequences

Mylip/Idol iRNA sequences were synthesized on a MerMade 192 synthesizerat 1 μmol scale.

For all the sequences in Table 5, ‘endolight’ chemistry was applied asdetailed below.

-   -   All pyrimidines (cytosine and uridine) in the sense strand        contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)    -   In the antisense strand, pyrimidines adjacent to (towards 5′        position) ribo A nucleoside were replaced with their        corresponding 2-O-Methyl nucleosides    -   A two base dTsdT extension at 3′ end of both sense and anti        sense sequences was introduced    -   The sequence file was converted to a text file to make it        compatible for loading in the MerMade 192 synthesis software

Synthesis, Cleavage and Deprotection

The synthesis of Mylip/Idol sequences used solid supportedoligonucleotide synthesis using phosphoramidite chemistry.

The synthesis of the above sequences was performed at 1 μm scale in 96well plates. The amidite solutions were prepared at 0.1M concentrationand ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.

The synthesized sequences were cleaved and deprotected in 96 wellplates, using methylamine in the first step and fluoride reagent in thesecond step. The crude sequences were precipitated using acetone:ethanol(80:20) mix and the pellets were re-suspended in 0.02M sodium acetatebuffer. Samples from each sequence were analyzed by LC-MS to confirm theidentity, UV for quantification, and a selected set of samples were alsoanalyzed by IEX chromatography to determine purity.

Purification and Desalting

All sequences were purified on AKTA explorer purification system usingSource 15Q column. Sample injection and collection was performed in 96well (1.8 mL-deep well) plates. A single peak corresponding to the fulllength sequence was collected in the eluent. The purified sequences weredesalted on a Sephadex G25 column using AKTA purifier. The desaltedMylip/Idol sequences were analyzed for concentration (by UV measurementat A260) and purity (by ion exchange HPLC). The single strands were thensubmitted for annealing.

Other embodiments are in the claims.

The invention claimed is:
 1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of Mylip/Idol, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a Mylip/Idol transcript which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:
 21. 2. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide.
 3. The dsRNA of claim 2, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide; a nucleotide comprising a 5′-phosphorothioate group; a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group; a 2′-deoxy-2′-fluoro modified nucleotide; a 2′-deoxy-modified nucleotide; a locked nucleotide; an abasic nucleotide; 2′-amino-modified nucleotide; 2′-alkyl-modified nucleotide; morpholino nucleotide; a phosphoramidate; and a non-natural base comprising nucleotide.
 4. The dsRNA of claim 1, wherein the region of complementarity is at least 17 nucleotides in length.
 5. The dsRNA of claim 1, wherein the region of complementarity is between 19 and 21 nucleotides in length.
 6. The dsRNA of claim 5, wherein the region of complementarity is 19 nucleotides in length.
 7. The dsRNA of claim 1, wherein each strand is no more than 30 nucleotides in length.
 8. The dsRNA of claim 1, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 9. The dsRNA of claim 1, further comprising a ligand.
 10. The dsRNA of claim 9, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
 11. A dsRNA for inhibiting the expression of Mylip/Idol, wherein the dsRNA comprises a sense strand consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 20, 22, 24, 26, 28, 32, and 34 and an antisense strand consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 21, 23, 25, 27, 29, 33, and
 35. 12. A pharmaceutical composition for inhibiting expression of a Mylip/Idol gene comprising a dsRNA comprising a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a Mylip/Idol transcript which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the sequence of SEQ ID NO:
 21. 13. The pharmaceutical composition of claim 12, further comprising a lipid formulation.
 14. The pharmaceutical composition of claim 13, wherein the lipid formulation is a SNALP, or XTC formulation.
 15. A method of treating a disorder mediated by Mylip/Idol expression comprising administering to a human in need of such treatment a therapeutically effective amount of a dsRNA comprising a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a Mylip/Idol transcript which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the sequence of SEQ ID NO: 21; or a pharmaceutical composition comprising a dsRNA comprising a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a Mylip/Idol transcript which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the sequence of SEQ ID NO:
 21. 16. The method of claim 15, wherein the human has a lipid disorder.
 17. The method of claim 15, wherein the human has a disorder associated with cholesterol metabolism.
 18. The method of claim 15, wherein the human has diabetes or atherosclerosis.
 19. The method of claim 17, wherein the administration of the dsRNA to the subject causes a decrease in Low Density Lipoprotein cholesterol (LDLc) in the serum of the subject by at least 10%.
 20. The method of claim 15, wherein the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
 21. The dsRNA of claim 1, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 20 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding antisense nucleotide sequence of SEQ ID NO:
 21. 22. The method of claim 15, wherein said dsRNA comprises at least one modified nucleotide.
 23. The method of claim 22, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2′-methyl modified nucleotide; a nucleotide comprising a 5′-phosphorothioate group; a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group; a 2′-deoxy-2′-fluoro modified nucleotide; a 2′-deoxy-modified nucleotide; a locked nucleotide; an abasic nucleotide; 2′-amino-modified nucleotide; 2′-alkyl-modified nucleotide; morpholino nucleotide; a phosphoramidate; and a non-natural base comprising nucleotide.
 24. The method of claim 15, wherein the region of complementarity is at least 17 nucleotides in length.
 25. The method of claim 15, wherein the region of complementarity is between 19 and 21 nucleotides in length.
 26. The method of claim 15, wherein the region of complementarity is 19 nucleotides in length.
 27. The method of claim 15, wherein each strand is no more than 30 nucleotides in length.
 28. The method of claim 15, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 29. The method of claim 15, wherein the dsRNA further comprises a ligand.
 30. The method of claim 29, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
 31. A method of treating a disorder mediated by Mylip/Idol expression comprising administering to a human in need of such treatment a therapeutically effective amount of a dsRNA comprising a sense strand consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 20, 22, 24, 26, 28, 32, and 34 and an antisense strand consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 21, 23, 25, 27, 29, 33, and
 35. 